1 Load pkgs

library(tidyverse)
## ── Attaching core tidyverse packages ──────────────────────── tidyverse 2.0.0 ──
## ✔ dplyr     1.1.3     ✔ readr     2.1.4
## ✔ forcats   1.0.0     ✔ stringr   1.5.0
## ✔ ggplot2   3.4.4     ✔ tibble    3.2.1
## ✔ lubridate 1.9.3     ✔ tidyr     1.3.0
## ✔ purrr     1.0.2     
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## ℹ Use the conflicted package (<http://conflicted.r-lib.org/>) to force all conflicts to become errors
library(here)
## here() starts at /Users/connor/Dropbox/SoyEndophytes/public_repo
library(patchwork)
library(cowplot)
## 
## Attaching package: 'cowplot'
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## The following object is masked from 'package:patchwork':
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##     align_plots
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##     stamp
library(ggVennDiagram)
## 
## Attaching package: 'ggVennDiagram'
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## The following object is masked from 'package:tidyr':
## 
##     unite
library(ggpubr)
## 
## Attaching package: 'ggpubr'
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## The following object is masked from 'package:cowplot':
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##     get_legend
library(vegan)
## Loading required package: permute
## Loading required package: lattice
## This is vegan 2.6-4
library(phyloseq)
## Warning: package 'phyloseq' was built under R version 4.2.2
library(SpiecEasi)
library(NetCoMi)
## 

2 Read in data

cleaned.files= here::here("datasets")
net.loc= here("network_analysis")
output.loc= here("outputs")

#its1
its1=read.csv(file= paste0(cleaned.files,"/cleaned.ASVs.ITS1.csv"), row.names = "X")

its1.net= read_csv(file= paste0(cleaned.files,"/cleaned.ASVs.ITS1.csv"))
## New names:
## Rows: 117 Columns: 423
## ── Column specification
## ──────────────────────────────────────────────────────── Delimiter: "," chr
## (1): ...1 dbl (422): Epicoccum dendrobii, Epicoccum dendrobii.1, Epicoccum
## thailandicu...
## ℹ Use `spec()` to retrieve the full column specification for this data. ℹ
## Specify the column types or set `show_col_types = FALSE` to quiet this message.
## • `` -> `...1`
#its2
its2 = read.csv(file= paste0(cleaned.files, "/cleaned.ASVs.its2.csv"), row.names = "X")

its2.net = read_csv(file= paste0(cleaned.files, "/cleaned.ASVs.its2.csv"))
## New names:
## Rows: 117 Columns: 1062
## ── Column specification
## ──────────────────────────────────────────────────────── Delimiter: "," chr
## (1): ...1 dbl (1061): Epicoccum sp., Cladosporium ramotenellum, Didymella sp.,
## Paracon...
## ℹ Use `spec()` to retrieve the full column specification for this data. ℹ
## Specify the column types or set `show_col_types = FALSE` to quiet this message.
## • `` -> `...1`
#bacteria
bac= read.csv(file= paste0(cleaned.files,"/cleaned.ASVs.bacteria.csv"), row.names = "X")

bac.net= read_csv(file= paste0(cleaned.files,"/cleaned.ASVs.bacteria.csv"))
## New names:
## Rows: 117 Columns: 214
## ── Column specification
## ──────────────────────────────────────────────────────── Delimiter: "," chr
## (1): ...1 dbl (213): Methylobacterium-Methylorubrum sp.,
## Methylobacterium-Methylorubru...
## ℹ Use `spec()` to retrieve the full column specification for this data. ℹ
## Specify the column types or set `show_col_types = FALSE` to quiet this message.
## • `` -> `...1`
# two additional ones for higher level taxa summaries
# bac.asvnos= read.csv(file= paste0(cleaned.files,"/cleaned.nothost.bacteria.asv_labeled_by_asv_no.csv"), row.names = "X")
# 
# tax dfs
bac.tax= read.csv(file= paste0(cleaned.files,"/cleaned.nothost.bacteria.taxa.matched_to_asv_no.csv"), row.names = "X")

its1.tax= read.csv(file= paste0(cleaned.files,"/cleaned.nothost.its1.taxa.matched_to_asv_no.csv"), row.names = "X")

its2.tax= read.csv(file= paste0(cleaned.files,"/cleaned.nothost.its2.taxa.matched_to_asv_no.csv"), row.names = "X")

#metadata
meta= read.csv(file= paste0(cleaned.files, "/cleaned.meta.csv"), sep=",", header = T)

2.1 Cleaning

# change plot names for congruence with metadata and taxa dfs
meta$site= sub("_", "-", meta$site)

its1= its1 %>% 
  rownames_to_column("site")

its1$site= sub("_", "-", its1$site)

its1 = its1 %>% 
  column_to_rownames("site")

meta.lite= meta %>% 
  dplyr::select(site, CO2, plot, rep)

# Clean up family names for more readable legend
its1.tax$Family.legend= gsub(".*__","",its1.tax$Family)

its2.tax$Family.legend= gsub(".*__","",its2.tax$Family)

# sample ID
# fix sample names
names(its1.net)[1] ="Sample"

its1.net$Sample= sub("_", "-", its1.net$Sample)

names(its2.net)[1] ="Sample"

names(bac.net)[1] ="Sample"

3 SUMMARY STATS

3.1 Read counts

# link meta to asv dfs too

## its1
read.counts.its1= its1 %>% 
  rownames_to_column("site") %>% 
  full_join(., meta.lite) %>% 
  dplyr::select(-c(plot, rep)) %>% 
  group_split(CO2)
## Joining with `by = join_by(site)`
names(read.counts.its1)<- c("control", "elevated")

read.counts.its1= map(read.counts.its1, dplyr::select,!CO2)

read.counts.its1= map(read.counts.its1, column_to_rownames, "site")

# filter out columns that are completely blank, these can be induced once the dataset is split by treatment
no.zeros.its1=list()
no.zeros.its1$control=read.counts.its1$control[colSums(read.counts.its1$control, na.rm = TRUE) > 0]
no.zeros.its1$elevated=read.counts.its1$elevated[colSums(read.counts.its1$elevated, na.rm = TRUE) > 0]

## bac
read.counts.bac= bac %>% 
  rownames_to_column("site") %>% 
  full_join(., meta.lite) %>% 
  dplyr::select(-c( plot, rep)) %>% 
  group_split(CO2)
## Joining with `by = join_by(site)`
names(read.counts.bac)<- c("control", "elevated")

read.counts.bac= map(read.counts.bac, dplyr::select,!CO2)

read.counts.bac= map(read.counts.bac, column_to_rownames, "site")


no.zeros.bac=list()
no.zeros.bac$control=read.counts.bac$control[colSums(read.counts.bac$control, na.rm = TRUE) > 0]
no.zeros.bac$elevated=read.counts.bac$elevated[colSums(read.counts.bac$elevated, na.rm = TRUE) > 0]

## its2
read.counts.its2= its2 %>% 
  rownames_to_column("site") %>% 
  full_join(., meta.lite) %>% 
  dplyr::select(-c(plot, rep)) %>% 
  group_split(CO2)
## Joining with `by = join_by(site)`
names(read.counts.its2)<- c("control", "elevated")

read.counts.its2= map(read.counts.its2, dplyr::select,!CO2)

read.counts.its2= map(read.counts.its2, column_to_rownames, "site")

map(read.counts.its2, sum)
## $control
## [1] 752887
## 
## $elevated
## [1] 964266
no.zeros.its2=list()
no.zeros.its2$control=read.counts.its2$control[colSums(read.counts.its2$control, na.rm = TRUE) > 0]
no.zeros.its2$elevated=read.counts.its2$elevated[colSums(read.counts.its2$elevated, na.rm = TRUE) > 0]
## sum read counts across primers 

its1.read.sums= map(no.zeros.its1, sum)
its2.read.sums= map(no.zeros.its2, sum)
bac.read.sums= map(no.zeros.bac, sum)

its1.read.sums.tot= sum(unlist(its1.read.sums, recursive=FALSE))
its2.read.sums.tot= sum(unlist(its2.read.sums, recursive=FALSE))
bac.read.sums.tot= sum(unlist(bac.read.sums, recursive=FALSE))

4 COMMUNITY METRICS

4.1 Richness and diversity

Per sample

#richness per sample
bac$richness= rowSums(bac > 0)

its1$richness= rowSums(its1 > 0)

its2$richness= rowSums(its2 > 0)

# shannon, simp

bac$H = diversity(bac)
its1$H = diversity(its1)
its2$H = diversity(its2)

bac$simp = diversity(bac, "simpson")
its1$simp = diversity(its1, "simpson")
its2$simp = diversity(its2, "simpson")

Per plot To calculate per plot richness we are going to have to do some extra massaging

plot.rich.bac= bac %>% 
  rownames_to_column("site")
plot.rich.its1= its1 %>% 
  rownames_to_column("site")
plot.rich.its2= its2 %>% 
  rownames_to_column("site")

plot.rich.bac=full_join(plot.rich.bac, meta.lite)
## Joining with `by = join_by(site)`
plot.rich.its1=full_join(plot.rich.its1, meta.lite)
## Joining with `by = join_by(site)`
plot.rich.its2=full_join(plot.rich.its2, meta.lite)
## Joining with `by = join_by(site)`

Link to metadata

# rownames to cols to link meta data in
bac.div= bac %>% 
  dplyr::select(richness, H, simp) %>% 
  rownames_to_column("site")

its1.div= its1 %>% 
  dplyr::select(richness, H, simp) %>% 
  rownames_to_column("site")

its2.div= its2 %>% 
  dplyr::select(richness, H, simp) %>% 
  rownames_to_column("site")


# link to meta
bac.div= full_join(bac.div, meta.lite)
## Joining with `by = join_by(site)`
its1.div= full_join(its1.div, meta.lite)
## Joining with `by = join_by(site)`
its2.div= full_join(its2.div, meta.lite)
## Joining with `by = join_by(site)`

4.2 Composition

Transform data to proportions or apply Hellinger

ITS1.asv.prop=prop.table(as.matrix(its1),1)

ITS1.asv.hell <- decostand(its1, "hellinger")

its2.asv.prop=prop.table(as.matrix(its2),1)

its2.asv.hell <- decostand(its2, "hellinger")

bacteria.asv.prop=prop.table(as.matrix(bac),1)

bacteria.asv.hell <- decostand(bac, "hellinger")

Visualization

NMDS for each primer on the transformed mat

ITS1

its1.mds <- metaMDS(ITS1.asv.prop)
## Run 0 stress 0.2066101 
## Run 1 stress 0.2313255 
## Run 2 stress 0.2180168 
## Run 3 stress 0.2017663 
## ... New best solution
## ... Procrustes: rmse 0.06273509  max resid 0.3785614 
## Run 4 stress 0.2107423 
## Run 5 stress 0.2005392 
## ... New best solution
## ... Procrustes: rmse 0.03079813  max resid 0.2249917 
## Run 6 stress 0.1999325 
## ... New best solution
## ... Procrustes: rmse 0.04999547  max resid 0.4502096 
## Run 7 stress 0.2022238 
## Run 8 stress 0.2150842 
## Run 9 stress 0.1997167 
## ... New best solution
## ... Procrustes: rmse 0.04094578  max resid 0.4052451 
## Run 10 stress 0.2043617 
## Run 11 stress 0.2065799 
## Run 12 stress 0.2040572 
## Run 13 stress 0.2157917 
## Run 14 stress 0.1987304 
## ... New best solution
## ... Procrustes: rmse 0.04639118  max resid 0.2758157 
## Run 15 stress 0.2412488 
## Run 16 stress 0.2033998 
## Run 17 stress 0.199623 
## Run 18 stress 0.2319903 
## Run 19 stress 0.2245278 
## Run 20 stress 0.23903 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      1: no. of iterations >= maxit
##     19: stress ratio > sratmax
its1.mds.mds.3 <- metaMDS(ITS1.asv.prop, k=3)
## Run 0 stress 0.1202522 
## Run 1 stress 0.1253933 
## Run 2 stress 0.1203019 
## ... Procrustes: rmse 0.005123648  max resid 0.04864147 
## Run 3 stress 0.1202526 
## ... Procrustes: rmse 0.0008150113  max resid 0.005710849 
## ... Similar to previous best
## Run 4 stress 0.1214577 
## Run 5 stress 0.1221056 
## Run 6 stress 0.1218708 
## Run 7 stress 0.1218712 
## Run 8 stress 0.1311642 
## Run 9 stress 0.1457618 
## Run 10 stress 0.1218723 
## Run 11 stress 0.1203025 
## ... Procrustes: rmse 0.005121691  max resid 0.04865503 
## Run 12 stress 0.1202528 
## ... Procrustes: rmse 0.0008590684  max resid 0.007162997 
## ... Similar to previous best
## Run 13 stress 0.1253963 
## Run 14 stress 0.122063 
## Run 15 stress 0.1221432 
## Run 16 stress 0.1221904 
## Run 17 stress 0.125394 
## Run 18 stress 0.120267 
## ... Procrustes: rmse 0.001957128  max resid 0.01780351 
## Run 19 stress 0.1203302 
## ... Procrustes: rmse 0.005250133  max resid 0.04853555 
## Run 20 stress 0.1504365 
## *** Best solution repeated 2 times
metaMDS(ITS1.asv.prop, k=3, previous.best = its1.mds.mds.3)
## Starting from 3-dimensional configuration
## Run 0 stress 0.1202522 
## Run 1 stress 0.1221793 
## Run 2 stress 0.1543107 
## Run 3 stress 0.1296756 
## Run 4 stress 0.1219415 
## Run 5 stress 0.1219415 
## Run 6 stress 0.1202594 
## ... Procrustes: rmse 0.00132137  max resid 0.0117324 
## Run 7 stress 0.1221693 
## Run 8 stress 0.1221717 
## Run 9 stress 0.1223207 
## Run 10 stress 0.1203027 
## ... Procrustes: rmse 0.005120812  max resid 0.04876093 
## Run 11 stress 0.127147 
## Run 12 stress 0.1202522 
## ... Procrustes: rmse 0.0003115428  max resid 0.002256778 
## ... Similar to previous best
## Run 13 stress 0.1308975 
## Run 14 stress 0.1219415 
## Run 15 stress 0.1219417 
## Run 16 stress 0.1270295 
## Run 17 stress 0.1282541 
## Run 18 stress 0.1274195 
## Run 19 stress 0.1202519 
## ... New best solution
## ... Procrustes: rmse 0.0006577628  max resid 0.004218151 
## ... Similar to previous best
## Run 20 stress 0.1288932 
## *** Best solution repeated 1 times
## 
## Call:
## metaMDS(comm = ITS1.asv.prop, k = 3, previous.best = its1.mds.mds.3) 
## 
## global Multidimensional Scaling using monoMDS
## 
## Data:     ITS1.asv.prop 
## Distance: bray 
## 
## Dimensions: 3 
## Stress:     0.1202519 
## Stress type 1, weak ties
## Best solution was repeated 1 time in 40 tries
## The best solution was from try 39 (random start)
## Scaling: centring, PC rotation, halfchange scaling 
## Species: expanded scores based on 'ITS1.asv.prop'
#hellinger
its1.mds.hell <- metaMDS(ITS1.asv.hell)
## Run 0 stress 0.2302418 
## Run 1 stress 0.2438084 
## Run 2 stress 0.2323092 
## Run 3 stress 0.2300196 
## ... New best solution
## ... Procrustes: rmse 0.00619494  max resid 0.06448405 
## Run 4 stress 0.2353881 
## Run 5 stress 0.2319402 
## Run 6 stress 0.2314957 
## Run 7 stress 0.25581 
## Run 8 stress 0.2309687 
## Run 9 stress 0.2301698 
## ... Procrustes: rmse 0.003744664  max resid 0.02944566 
## Run 10 stress 0.2376224 
## Run 11 stress 0.2455472 
## Run 12 stress 0.2317653 
## Run 13 stress 0.2382812 
## Run 14 stress 0.2307375 
## Run 15 stress 0.2300196 
## ... New best solution
## ... Procrustes: rmse 0.0005442131  max resid 0.00422123 
## ... Similar to previous best
## Run 16 stress 0.2315796 
## Run 17 stress 0.230111 
## ... Procrustes: rmse 0.002907652  max resid 0.0253345 
## Run 18 stress 0.2308776 
## Run 19 stress 0.4134527 
## Run 20 stress 0.2379424 
## *** Best solution repeated 1 times
its1.mds.3.hell <- metaMDS(ITS1.asv.hell, k=3)
## Run 0 stress 0.1632476 
## Run 1 stress 0.1632741 
## ... Procrustes: rmse 0.0268868  max resid 0.268996 
## Run 2 stress 0.1632468 
## ... New best solution
## ... Procrustes: rmse 0.000177154  max resid 0.001699588 
## ... Similar to previous best
## Run 3 stress 0.1632461 
## ... New best solution
## ... Procrustes: rmse 0.0001052523  max resid 0.0007078326 
## ... Similar to previous best
## Run 4 stress 0.1632543 
## ... Procrustes: rmse 0.004041176  max resid 0.03915495 
## Run 5 stress 0.1640441 
## Run 6 stress 0.1632474 
## ... Procrustes: rmse 0.003151712  max resid 0.03056742 
## Run 7 stress 0.1662856 
## Run 8 stress 0.1632748 
## ... Procrustes: rmse 0.02738609  max resid 0.274164 
## Run 9 stress 0.1639667 
## Run 10 stress 0.166668 
## Run 11 stress 0.1632662 
## ... Procrustes: rmse 0.00500943  max resid 0.04860608 
## Run 12 stress 0.1632597 
## ... Procrustes: rmse 0.004487319  max resid 0.04326371 
## Run 13 stress 0.1632487 
## ... Procrustes: rmse 0.0007699863  max resid 0.004556935 
## ... Similar to previous best
## Run 14 stress 0.1632756 
## ... Procrustes: rmse 0.005761526  max resid 0.05594761 
## Run 15 stress 0.1647938 
## Run 16 stress 0.1647923 
## Run 17 stress 0.1632744 
## ... Procrustes: rmse 0.02725636  max resid 0.2729225 
## Run 18 stress 0.1641238 
## Run 19 stress 0.1648895 
## Run 20 stress 0.1659173 
## *** Best solution repeated 2 times
metaMDS(ITS1.asv.hell, k=3, previous.best = its1.mds.3.hell)
## Starting from 3-dimensional configuration
## Run 0 stress 0.1632461 
## Run 1 stress 0.164793 
## Run 2 stress 0.1632471 
## ... Procrustes: rmse 0.0001806698  max resid 0.001622254 
## ... Similar to previous best
## Run 3 stress 0.1648136 
## Run 4 stress 0.164887 
## Run 5 stress 0.1633711 
## ... Procrustes: rmse 0.01036408  max resid 0.1009508 
## Run 6 stress 0.1633175 
## ... Procrustes: rmse 0.008097086  max resid 0.07872191 
## Run 7 stress 0.163263 
## ... Procrustes: rmse 0.004731158  max resid 0.04557621 
## Run 8 stress 0.1634559 
## ... Procrustes: rmse 0.007804179  max resid 0.06323481 
## Run 9 stress 0.1632745 
## ... Procrustes: rmse 0.02700637  max resid 0.2702803 
## Run 10 stress 0.1632426 
## ... New best solution
## ... Procrustes: rmse 0.001449912  max resid 0.01408879 
## Run 11 stress 0.1632488 
## ... Procrustes: rmse 0.001910242  max resid 0.01837757 
## Run 12 stress 0.163248 
## ... Procrustes: rmse 0.001631157  max resid 0.01580063 
## Run 13 stress 0.1653427 
## Run 14 stress 0.1632739 
## ... Procrustes: rmse 0.02834993  max resid 0.2845359 
## Run 15 stress 0.1632468 
## ... Procrustes: rmse 0.001582545  max resid 0.01518116 
## Run 16 stress 0.1632749 
## ... Procrustes: rmse 0.0285043  max resid 0.2860614 
## Run 17 stress 0.1652667 
## Run 18 stress 0.1645012 
## Run 19 stress 0.1632556 
## ... Procrustes: rmse 0.002736291  max resid 0.02640758 
## Run 20 stress 0.1632751 
## ... Procrustes: rmse 0.0284876  max resid 0.2858805 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     10: no. of iterations >= maxit
##     10: stress ratio > sratmax
## 
## Call:
## metaMDS(comm = ITS1.asv.hell, k = 3, previous.best = its1.mds.3.hell) 
## 
## global Multidimensional Scaling using monoMDS
## 
## Data:     ITS1.asv.hell 
## Distance: bray 
## 
## Dimensions: 3 
## Stress:     0.1632426 
## Stress type 1, weak ties
## Best solution was not repeated after 40 tries
## The best solution was from try 30 (random start)
## Scaling: centring, PC rotation, halfchange scaling 
## Species: expanded scores based on 'ITS1.asv.hell'

A three dim k=3 solution is better

nmds.its1= its1.mds.mds.3 ## CHANGE ME
#site scores
data.scores.its1 <- as.data.frame(scores(nmds.its1)$sites)  #Using the scores function from vegan to extract the site scores and convert to a data.frame
data.scores.its1$site <- rownames(data.scores.its1)  # create a column of site names, from the rownames of data.scores
data.scores.its1=full_join(data.scores.its1, meta)
## Joining with `by = join_by(site)`
#species scores
species.scores.its1 <- as.data.frame(scores(nmds.its1, "species"))  #Using the scores function from vegan to extract the species scores and convert to a data.frame
species.scores.its1$species <- rownames(species.scores.its1)  # create a column of species, from the rownames of species.scores
its1.A= ggplot(data= data.scores.its1,aes(x=NMDS1,y=NMDS2,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.its1,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.its1,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
## Warning: The `<scale>` argument of `guides()` cannot be `FALSE`. Use "none" instead as
## of ggplot2 3.3.4.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
if("NMDS3" %in% colnames(data.scores.its1) ==TRUE){
its1.B=ggplot(data= data.scores.its1,aes(x=NMDS1,y=NMDS3,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.its1,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.its1,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
}

#axis 2 vs 3
if("NMDS3" %in% colnames(data.scores.its1) ==TRUE){
its1.C=ggplot(data= data.scores.its1,aes(x=NMDS2,y=NMDS3,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.its1,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.its1,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
}

its1.all= its1.A + its1.B + its1.C

its2

its2.mds.hell <- metaMDS(its2.asv.hell)
## Run 0 stress 0.2371546 
## Run 1 stress 0.2468062 
## Run 2 stress 0.2434688 
## Run 3 stress 0.2401786 
## Run 4 stress 0.2472459 
## Run 5 stress 0.254371 
## Run 6 stress 0.2426449 
## Run 7 stress 0.2402574 
## Run 8 stress 0.2437059 
## Run 9 stress 0.2434794 
## Run 10 stress 0.244101 
## Run 11 stress 0.2565453 
## Run 12 stress 0.2464406 
## Run 13 stress 0.2396452 
## Run 14 stress 0.2519282 
## Run 15 stress 0.2484724 
## Run 16 stress 0.2416936 
## Run 17 stress 0.2461888 
## Run 18 stress 0.2468827 
## Run 19 stress 0.2487219 
## Run 20 stress 0.2502629 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      7: no. of iterations >= maxit
##     13: stress ratio > sratmax
its2.mds.hell.3 <- metaMDS(its2.asv.hell, k=3)
## Run 0 stress 0.1795205 
## Run 1 stress 0.1911154 
## Run 2 stress 0.1833896 
## Run 3 stress 0.1859847 
## Run 4 stress 0.1808877 
## Run 5 stress 0.1876924 
## Run 6 stress 0.182177 
## Run 7 stress 0.1911371 
## Run 8 stress 0.1803418 
## Run 9 stress 0.1811293 
## Run 10 stress 0.1795707 
## ... Procrustes: rmse 0.00636268  max resid 0.06330372 
## Run 11 stress 0.1797572 
## ... Procrustes: rmse 0.01798144  max resid 0.1116487 
## Run 12 stress 0.1888051 
## Run 13 stress 0.1840194 
## Run 14 stress 0.1811201 
## Run 15 stress 0.1804232 
## Run 16 stress 0.1796063 
## ... Procrustes: rmse 0.01256045  max resid 0.1082769 
## Run 17 stress 0.1806517 
## Run 18 stress 0.1832335 
## Run 19 stress 0.1804368 
## Run 20 stress 0.1853824 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     18: no. of iterations >= maxit
##      2: stress ratio > sratmax
metaMDS(its2.asv.hell, k=3, previous.best = its2.mds.hell.3)
## Starting from 3-dimensional configuration
## Run 0 stress 0.1795205 
## Run 1 stress 0.1827519 
## Run 2 stress 0.1798022 
## ... Procrustes: rmse 0.01582041  max resid 0.09598276 
## Run 3 stress 0.1803149 
## Run 4 stress 0.1848867 
## Run 5 stress 0.1819429 
## Run 6 stress 0.1796089 
## ... Procrustes: rmse 0.0126113  max resid 0.1046857 
## Run 7 stress 0.1803114 
## Run 8 stress 0.1796505 
## ... Procrustes: rmse 0.01363958  max resid 0.1047757 
## Run 9 stress 0.1795696 
## ... Procrustes: rmse 0.006330195  max resid 0.06301324 
## Run 10 stress 0.1837267 
## Run 11 stress 0.1841725 
## Run 12 stress 0.1850958 
## Run 13 stress 0.1876756 
## Run 14 stress 0.1827281 
## Run 15 stress 0.1802518 
## Run 16 stress 0.1849083 
## Run 17 stress 0.1819602 
## Run 18 stress 0.1846738 
## Run 19 stress 0.1801131 
## Run 20 stress 0.1822352 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     18: no. of iterations >= maxit
##      2: stress ratio > sratmax
## 
## Call:
## metaMDS(comm = its2.asv.hell, k = 3, previous.best = its2.mds.hell.3) 
## 
## global Multidimensional Scaling using monoMDS
## 
## Data:     its2.asv.hell 
## Distance: bray 
## 
## Dimensions: 3 
## Stress:     0.1795205 
## Stress type 1, weak ties
## Best solution was not repeated after 40 tries
## The best solution was from try 0 (metric scaling or null solution)
## Scaling: centring, PC rotation, halfchange scaling 
## Species: expanded scores based on 'its2.asv.hell'
its2.mds.prop <- metaMDS(its2.asv.prop)
## Run 0 stress 0.2014211 
## Run 1 stress 0.2107384 
## Run 2 stress 0.2096602 
## Run 3 stress 0.2135925 
## Run 4 stress 0.2086454 
## Run 5 stress 0.2154752 
## Run 6 stress 0.2125952 
## Run 7 stress 0.2060756 
## Run 8 stress 0.2050126 
## Run 9 stress 0.209379 
## Run 10 stress 0.2165897 
## Run 11 stress 0.2087148 
## Run 12 stress 0.2091691 
## Run 13 stress 0.2155498 
## Run 14 stress 0.223542 
## Run 15 stress 0.2089512 
## Run 16 stress 0.2076853 
## Run 17 stress 0.2071709 
## Run 18 stress 0.2181931 
## Run 19 stress 0.2086439 
## Run 20 stress 0.2098481 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      7: no. of iterations >= maxit
##     13: stress ratio > sratmax
its2.mds.prop.3 <- metaMDS(its2.asv.prop, k=3)
## Run 0 stress 0.1501586 
## Run 1 stress 0.1499261 
## ... New best solution
## ... Procrustes: rmse 0.04452019  max resid 0.2160773 
## Run 2 stress 0.1501838 
## ... Procrustes: rmse 0.04517297  max resid 0.2398887 
## Run 3 stress 0.1506645 
## Run 4 stress 0.150633 
## Run 5 stress 0.1496062 
## ... New best solution
## ... Procrustes: rmse 0.02365725  max resid 0.2072309 
## Run 6 stress 0.1506949 
## Run 7 stress 0.1499125 
## ... Procrustes: rmse 0.04418184  max resid 0.239235 
## Run 8 stress 0.1535526 
## Run 9 stress 0.1547474 
## Run 10 stress 0.1542647 
## Run 11 stress 0.1503214 
## Run 12 stress 0.1562014 
## Run 13 stress 0.1529133 
## Run 14 stress 0.1520036 
## Run 15 stress 0.150364 
## Run 16 stress 0.1536149 
## Run 17 stress 0.1506543 
## Run 18 stress 0.1534635 
## Run 19 stress 0.1570662 
## Run 20 stress 0.1543156 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     15: no. of iterations >= maxit
##      5: stress ratio > sratmax
nmds.its2=metaMDS(its2.asv.prop, k=3, previous.best = its2.mds.prop.3)
## Starting from 3-dimensional configuration
## Run 0 stress 0.1496062 
## Run 1 stress 0.1499121 
## ... Procrustes: rmse 0.03875206  max resid 0.2443582 
## Run 2 stress 0.1542449 
## Run 3 stress 0.1506679 
## Run 4 stress 0.151191 
## Run 5 stress 0.1546472 
## Run 6 stress 0.1541318 
## Run 7 stress 0.1502279 
## Run 8 stress 0.1508227 
## Run 9 stress 0.1553195 
## Run 10 stress 0.1496495 
## ... Procrustes: rmse 0.003538382  max resid 0.03443763 
## Run 11 stress 0.1509365 
## Run 12 stress 0.1511187 
## Run 13 stress 0.1533337 
## Run 14 stress 0.1520943 
## Run 15 stress 0.1513405 
## Run 16 stress 0.1504105 
## Run 17 stress 0.1513908 
## Run 18 stress 0.1508853 
## Run 19 stress 0.1516135 
## Run 20 stress 0.1510949 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     17: no. of iterations >= maxit
##      3: stress ratio > sratmax
#site scores
data.scores.its2 <- as.data.frame(scores(nmds.its2)$sites)  #Using the scores function from vegan to extract the site scores and convert to a data.frame
data.scores.its2$site <- rownames(data.scores.its2)  # create a column of site names, from the rownames of data.scores

data.scores.its2=full_join(data.scores.its2, meta)
## Joining with `by = join_by(site)`
#species scores
species.scores.its2 <- as.data.frame(scores(nmds.its2, "species"))  #Using the scores function from vegan to extract the species scores and convert to a data.frame
species.scores.its2$species <- rownames(species.scores.its2)  # create a column of species, from the rownames of species.scores
its2.A=ggplot(data= data.scores.its2,aes(x=NMDS1,y=NMDS2,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.its2,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.its2,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)

#axis 1 v 3

if("NMDS3" %in% colnames(data.scores.its2) ==TRUE){
its2.B=ggplot(data= data.scores.its2,aes(x=NMDS1,y=NMDS3,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.its2,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.its2,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
}

#axis 2 vs 3
if("NMDS3" %in% colnames(data.scores.its2) ==TRUE){
its2.C=ggplot(data= data.scores.its2,aes(x=NMDS2,y=NMDS3,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.its2,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.its2,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
}

its2.all= its2.A + its2.B + its2.C

Bacteria

# NMDS

bacteria.mds.hell <- metaMDS(bacteria.asv.hell)
## Run 0 stress 0.2129363 
## Run 1 stress 0.2331149 
## Run 2 stress 0.2365677 
## Run 3 stress 0.2249617 
## Run 4 stress 0.2205346 
## Run 5 stress 0.2220319 
## Run 6 stress 0.2113902 
## ... New best solution
## ... Procrustes: rmse 0.02221691  max resid 0.1556173 
## Run 7 stress 0.2168891 
## Run 8 stress 0.2211196 
## Run 9 stress 0.2130616 
## Run 10 stress 0.2305469 
## Run 11 stress 0.229972 
## Run 12 stress 0.2302245 
## Run 13 stress 0.2254145 
## Run 14 stress 0.2295191 
## Run 15 stress 0.219298 
## Run 16 stress 0.2118241 
## ... Procrustes: rmse 0.01711956  max resid 0.1641574 
## Run 17 stress 0.2347548 
## Run 18 stress 0.2113892 
## ... New best solution
## ... Procrustes: rmse 0.005853489  max resid 0.06050097 
## Run 19 stress 0.2193599 
## Run 20 stress 0.2113683 
## ... New best solution
## ... Procrustes: rmse 0.005833782  max resid 0.06067458 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      2: no. of iterations >= maxit
##     18: stress ratio > sratmax
bacteria.mds.hell.3 <- metaMDS(bacteria.asv.hell, k=3)
## Run 0 stress 0.1579925 
## Run 1 stress 0.158922 
## Run 2 stress 0.1578357 
## ... New best solution
## ... Procrustes: rmse 0.02803642  max resid 0.1370321 
## Run 3 stress 0.1579638 
## ... Procrustes: rmse 0.04761142  max resid 0.1523802 
## Run 4 stress 0.1604293 
## Run 5 stress 0.1575072 
## ... New best solution
## ... Procrustes: rmse 0.04638111  max resid 0.1543406 
## Run 6 stress 0.1583354 
## Run 7 stress 0.1581992 
## Run 8 stress 0.1588536 
## Run 9 stress 0.158729 
## Run 10 stress 0.1573232 
## ... New best solution
## ... Procrustes: rmse 0.01089757  max resid 0.0766522 
## Run 11 stress 0.1579875 
## Run 12 stress 0.1583026 
## Run 13 stress 0.157214 
## ... New best solution
## ... Procrustes: rmse 0.04014039  max resid 0.1455551 
## Run 14 stress 0.1578314 
## Run 15 stress 0.1618956 
## Run 16 stress 0.160168 
## Run 17 stress 0.1598794 
## Run 18 stress 0.1600597 
## Run 19 stress 0.1572211 
## ... Procrustes: rmse 0.04075845  max resid 0.1470878 
## Run 20 stress 0.1619899 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      5: no. of iterations >= maxit
##     15: stress ratio > sratmax
metaMDS(bacteria.asv.hell, k=3, previous.best = bacteria.mds.hell.3)
## Starting from 3-dimensional configuration
## Run 0 stress 0.157214 
## Run 1 stress 0.159564 
## Run 2 stress 0.1585037 
## Run 3 stress 0.1594298 
## Run 4 stress 0.1570948 
## ... New best solution
## ... Procrustes: rmse 0.007329505  max resid 0.06790744 
## Run 5 stress 0.1580529 
## Run 6 stress 0.1578176 
## Run 7 stress 0.1579348 
## Run 8 stress 0.1584385 
## Run 9 stress 0.1600199 
## Run 10 stress 0.1578467 
## Run 11 stress 0.1571313 
## ... Procrustes: rmse 0.00178704  max resid 0.01595673 
## Run 12 stress 0.1573814 
## ... Procrustes: rmse 0.0396866  max resid 0.1452538 
## Run 13 stress 0.1589413 
## Run 14 stress 0.1590745 
## Run 15 stress 0.1572071 
## ... Procrustes: rmse 0.00673171  max resid 0.06833147 
## Run 16 stress 0.157541 
## ... Procrustes: rmse 0.02575474  max resid 0.1366501 
## Run 17 stress 0.1600043 
## Run 18 stress 0.1579214 
## Run 19 stress 0.1628412 
## Run 20 stress 0.1581247 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      7: no. of iterations >= maxit
##     13: stress ratio > sratmax
## 
## Call:
## metaMDS(comm = bacteria.asv.hell, k = 3, previous.best = bacteria.mds.hell.3) 
## 
## global Multidimensional Scaling using monoMDS
## 
## Data:     bacteria.asv.hell 
## Distance: bray 
## 
## Dimensions: 3 
## Stress:     0.1570948 
## Stress type 1, weak ties
## Best solution was not repeated after 40 tries
## The best solution was from try 24 (random start)
## Scaling: centring, PC rotation, halfchange scaling 
## Species: expanded scores based on 'bacteria.asv.hell'
bacteria.mds.prop <- metaMDS(bacteria.asv.prop)
## Run 0 stress 0.1627288 
## Run 1 stress 0.1782991 
## Run 2 stress 0.1682278 
## Run 3 stress 0.1755529 
## Run 4 stress 0.178149 
## Run 5 stress 0.1730015 
## Run 6 stress 0.1756586 
## Run 7 stress 0.1698679 
## Run 8 stress 0.1667112 
## Run 9 stress 0.1794483 
## Run 10 stress 0.1765297 
## Run 11 stress 0.1689898 
## Run 12 stress 0.182672 
## Run 13 stress 0.1731982 
## Run 14 stress 0.171074 
## Run 15 stress 0.1730032 
## Run 16 stress 0.1765308 
## Run 17 stress 0.1629908 
## ... Procrustes: rmse 0.04530894  max resid 0.4663444 
## Run 18 stress 0.1775802 
## Run 19 stress 0.1775409 
## Run 20 stress 0.1766715 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     20: stress ratio > sratmax
bacteria.mds.prop.3 <- metaMDS(bacteria.asv.prop, k=3)
## Run 0 stress 0.1129494 
## Run 1 stress 0.1125481 
## ... New best solution
## ... Procrustes: rmse 0.04755482  max resid 0.2724473 
## Run 2 stress 0.1130145 
## ... Procrustes: rmse 0.04416979  max resid 0.2683824 
## Run 3 stress 0.112425 
## ... New best solution
## ... Procrustes: rmse 0.05194885  max resid 0.2596961 
## Run 4 stress 0.1121783 
## ... New best solution
## ... Procrustes: rmse 0.02642323  max resid 0.1802529 
## Run 5 stress 0.1115598 
## ... New best solution
## ... Procrustes: rmse 0.04034403  max resid 0.1902641 
## Run 6 stress 0.113141 
## Run 7 stress 0.1135761 
## Run 8 stress 0.1138553 
## Run 9 stress 0.1134738 
## Run 10 stress 0.1140286 
## Run 11 stress 0.1118905 
## ... Procrustes: rmse 0.04234992  max resid 0.2694442 
## Run 12 stress 0.1112987 
## ... New best solution
## ... Procrustes: rmse 0.01146419  max resid 0.11546 
## Run 13 stress 0.1116482 
## ... Procrustes: rmse 0.0352086  max resid 0.1347775 
## Run 14 stress 0.1189774 
## Run 15 stress 0.1196039 
## Run 16 stress 0.1143562 
## Run 17 stress 0.1143646 
## Run 18 stress 0.1122263 
## Run 19 stress 0.1124548 
## Run 20 stress 0.1137566 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##     16: no. of iterations >= maxit
##      4: stress ratio > sratmax
nmds.bac=metaMDS(bacteria.asv.hell, k=3, previous.best = bacteria.mds.hell.3)
## Starting from 3-dimensional configuration
## Run 0 stress 0.157214 
## Run 1 stress 0.1574495 
## ... Procrustes: rmse 0.04249269  max resid 0.1481377 
## Run 2 stress 0.1585958 
## Run 3 stress 0.1582582 
## Run 4 stress 0.1583351 
## Run 5 stress 0.1595911 
## Run 6 stress 0.1579867 
## Run 7 stress 0.1579312 
## Run 8 stress 0.1581083 
## Run 9 stress 0.1572211 
## ... Procrustes: rmse 0.0407458  max resid 0.1472467 
## Run 10 stress 0.1579289 
## Run 11 stress 0.1590843 
## Run 12 stress 0.1574612 
## ... Procrustes: rmse 0.04243223  max resid 0.148298 
## Run 13 stress 0.1599498 
## Run 14 stress 0.1587643 
## Run 15 stress 0.1598681 
## Run 16 stress 0.1581038 
## Run 17 stress 0.157769 
## Run 18 stress 0.1579328 
## Run 19 stress 0.1582438 
## Run 20 stress 0.157221 
## ... Procrustes: rmse 0.04076249  max resid 0.1470826 
## *** Best solution was not repeated -- monoMDS stopping criteria:
##      5: no. of iterations >= maxit
##     15: stress ratio > sratmax
  # no convergence k=2
#site scores
data.scores.bac <- as.data.frame(scores(nmds.bac)$sites)  #Using the scores function from vegan to extract the site scores and convert to a data.frame
data.scores.bac$site <- rownames(data.scores.bac)  # create a column of site names, from the rownames of data.scores

data.scores.bac=full_join(data.scores.bac, meta)
## Joining with `by = join_by(site)`
#species scores
species.scores.bac <- as.data.frame(scores(nmds.bac, "species"))  #Using the scores function from vegan to extract the species scores and convert to a data.frame
species.scores.bac$species <- rownames(species.scores.bac)  # create a column of species, from the rownames of species.scores
bac.a=ggplot(data= data.scores.bac,aes(x=NMDS1,y=NMDS2,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.bac,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.bac,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue"), labels= c("Elevated", "Ambient")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)

#axis 1 v 3

if("NMDS3" %in% colnames(data.scores.bac) ==TRUE){
bac.b=ggplot(data= data.scores.bac,aes(x=NMDS1,y=NMDS3,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.bac,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.bac,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
}

#axis 2 vs 3
if("NMDS3" %in% colnames(data.scores.bac) ==TRUE){
bac.c= ggplot(data= data.scores.bac,aes(x=NMDS2,y=NMDS3,shape= CO2,colour=CO2)) + 
  #geom_text(data=species.scores.bac,aes(x=NMDS1,y=NMDS2,label=species),alpha=0.5) +  # add the species labels
  geom_point(size=3) + # add the point markers
  #geom_text(data=data.scores.bac,aes(x=NMDS1,y=NMDS2,label=site),size=6,vjust=0) +  # add the site labels
  scale_colour_manual(values=c("Elevated" = "red", "Control" = "blue")) +
  coord_equal() +
  theme_bw()+
  guides(color=guide_legend(expression(paste("CO"["2"]))), shape = FALSE)
}

bac.all= bac.a + bac.b + bac.c

Fig 1: multiplot fungi and bacterial

its2.A.noleg= its2.A + theme(legend.position = "none") + geom_label(aes(x = 0.3, y = 1.5, label = "Fungal composition"), inherit.aes = F)

bac.a.bot= bac.a + theme(legend.position = "bottom") + geom_label(aes(x = -.2, y = 1, label = "Bacterial composition"), inherit.aes = F) + expand_limits(x = 0.7, y= 1.2)

fig1.vertical= its2.A.noleg / bac.a.bot 

ggsave(paste0(output.loc, "/fig.1.pdf"), fig1.vertical, width = 6)
## Saving 6 x 5 in image
ggsave(paste0(output.loc, "/fig.1.jpg"), fig1.vertical, width = 6)
## Saving 6 x 5 in image

4.2.1 Stats

ITS1

# plot 20_9 doesn't appear to be in the ASV tab and is blank in the meta tab
meta.fin=meta %>% 
  filter(site !="20_9")
adonmod=adonis2(ITS1.asv.hell ~ CO2, data = meta.fin)


# check for dispersion
  #use data.scores df to get corresponding treatment assignments in proper order
treatment= data.scores.its1 %>% 
  filter(site!= "20_9")

dis.its1= vegdist(ITS1.asv.hell)
mod <- betadisper(dis.its1, treatment$CO2)
anova(mod)
(mod.HSD <- TukeyHSD(mod))
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = distances ~ group, data = df)
## 
## $group
##                         diff         lwr        upr    p adj
## Elevated-Control 0.007349717 -0.02828053 0.04297996 0.683596
plot(mod.HSD)

its2

adonmod_ITS2=adonis2(its2.asv.prop ~ CO2, data = meta.fin)

# check for dispersion
  #use data.scores df to get corresponding treatment assignments in proper order
treatment= data.scores.its2 %>% 
  filter(site!= "20_9")

dis= vegdist(its2.asv.prop)
mod <- betadisper(dis, treatment$CO2)
anova(mod)
(mod.HSD <- TukeyHSD(mod))
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = distances ~ group, data = df)
## 
## $group
##                         diff         lwr        upr     p adj
## Elevated-Control 0.009565556 -0.03537924 0.05451035 0.6741235
plot(mod.HSD)

Bacteria

# adonis
adonmod_16s=adonis2(bacteria.asv.hell ~ CO2, data = meta.fin)

# check for dispersion
  #use data.scores df to get corresponding treatment assignments in proper order
treatment= data.scores.bac %>% 
  filter(site!= "20-9")

dis= vegdist(bacteria.asv.hell)
mod <- betadisper(dis, treatment$CO2)
anova(mod)
(mod.HSD <- TukeyHSD(mod))
##   Tukey multiple comparisons of means
##     95% family-wise confidence level
## 
## Fit: aov(formula = distances ~ group, data = df)
## 
## $group
##                        diff         lwr        upr     p adj
## Elevated-Control 0.01786205 -0.02857368 0.06429777 0.4476551
plot(mod.HSD)

5 NETWORKS

  • Filtering low abundant ASVs, maintaining ASVs with more than 10 read counts

Remove low abundant ASVs

# ITS1
 its1.net= its1.net %>% column_to_rownames("Sample")
its1.sub=its1.net[ ,colSums(its1.net[,1:ncol(its1.net)]) >= 10]
paste("filtering low abundant ASVs removes", ncol(its1.net)- ncol(its1.sub), "ASVs")
## [1] "filtering low abundant ASVs removes 25 ASVs"
# its2
its2.net= its2.net %>% column_to_rownames("Sample")
its2.sub=its2.net[ ,colSums(its2.net[,1:ncol(its2.net)]) >= 10]
paste("filtering low abundant ASVs removes", ncol(its2.net)- ncol(its2.sub), "ASVs")
## [1] "filtering low abundant ASVs removes 36 ASVs"
# 16s
bac.net= bac.net %>% column_to_rownames("Sample")
bac.sub=bac.net[ ,colSums(bac.net[,2:ncol(bac.net)]) >= 10]
paste("filtering low abundant ASVs removes", ncol(bac.net)- ncol(bac.sub), "ASVs")
## [1] "filtering low abundant ASVs removes 23 ASVs"

Filter all.tax by the filtered asv tables

its1.sub.vec= names(its1.sub)

its1.tax.sub= its1.tax %>% 
  filter(uniqueID %in% its1.sub.vec)

its2.sub.vec= names(its2.sub)

its2.tax.sub= its2.tax %>% 
  filter(uniqueID %in% its2.sub.vec)

bac.sub.vec= names(bac.sub)

bac.tax.sub= bac.tax %>% 
  filter(uniqueID %in% bac.sub.vec)

Split Elevated CO2 and Ambient

Combine 3 asv dfs into a list item

# all.asvs= list(ITS1.asv.filter, its2.asv.filter, bacteria.asv.tab.filter) #old
all.asvs= list(its1.sub, its2.sub, bac.sub)
names(all.asvs)<- c("its1", "its2", "16s")

# all.tax= list(its1.tax, its2.tax, bac.tax) #old
all.tax= list(its1.tax.sub, its2.tax.sub, bac.tax.sub)

names(all.tax)<- c("its1", "its2", "16s")
# make a col named sample to link metadata in
all.asvs=all.asvs %>% 
map(rownames_to_column, "Sample")

# add metadata 
  
meta$site= sub("_", "-", meta$site)

meta.lite= meta %>% 
  select(site, CO2) %>% 
  rename(Sample= site)

all.asvs= all.asvs %>% 
  map(full_join, meta.lite) %>% # add metadata to asv data so we know which Samples are Elevated and which are Control
  map(relocate, "CO2", .after= "Sample") # move co2 treatment to start of df for each so we can see it
## Joining with `by = join_by(Sample)`
## Joining with `by = join_by(Sample)`
## Joining with `by = join_by(Sample)`
Elevated=all.asvs %>% 
  map(filter, CO2 == "Elevated") %>% 
  map(select, -c(CO2)) %>% 
  map( column_to_rownames, "Sample")

# Elevated[1] # check to see if that worked
Elevated[3]
## $`16s`
##       Methylobacterium-Methylorubrum sp. Methylobacterium-Methylorubrum sp..1
## 20-10                               2315                                    9
## 20-11                                540                                    0
## 20-12                                324                                    0
## 20-13                                144                                  715
## 20-14                                 19                                 1291
## 20-15                                289                                    0
## 20-2                                1123                                    6
## 20-3                                3957                                  546
## 20-4                                2433                                  419
## 20-5                                3374                                    0
## 20-6                                 949                                  196
## 20-7                                1171                                    0
## 20-8                                 229                                   96
## 21-1                                   0                                    0
## 21-10                               2357                                    0
## 21-11                                728                                  365
## 21-12                                  0                                  433
## 21-13                               1119                                  153
## 21-14                               1401                                    0
## 21-15                                  0                                    0
## 21-2                                   0                                   12
## 21-3                                1027                                    0
## 21-4                                1728                                    0
## 21-5                                2125                                    0
## 21-6                                 532                                  170
## 21-7                                2534                                    0
## 21-8                                 539                                    0
## 21-9                                  46                                    5
## 28-1                                  14                                   80
## 28-10                                625                                   16
## 28-11                                851                                 1008
## 28-12                               1987                                    8
## 28-13                               3117                                    0
## 28-14                               4430                                    0
## 28-15                               2629                                  286
## 28-2                                1622                                  139
## 28-3                                2263                                    0
## 28-4                                1257                                    0
## 28-5                                  72                                  208
## 28-6                                2938                                   24
## 28-7                                 956                                  131
## 28-8                                 840                                    0
## 28-9                                3037                                    0
## 29-1                                2265                                  572
## 29-10                                  0                                 1316
## 29-11                               3637                                    0
## 29-12                                237                                  853
## 29-13                               1664                                 2219
## 29-14                               4051                                  356
## 29-15                                 38                                  822
## 29-2                                1553                                   37
## 29-3                                 785                                    0
## 29-4                                1749                                    0
## 29-5                                  29                                  629
## 29-6                                1574                                  311
## 29-7                                1814                                 3066
## 29-8                                 910                                  342
## 29-9                                 301                                    0
##       Sphingomonas cynarae Aureimonas sp. Escherichia-Shigella sp.
## 20-10                 1823             50                        0
## 20-11                    0             86                        0
## 20-12                    0              0                        0
## 20-13                    0              0                        0
## 20-14                  102             14                        0
## 20-15                    0              0                        0
## 20-2                   209            276                        0
## 20-3                     0             16                        0
## 20-4                     0              0                        0
## 20-5                   340              0                      156
## 20-6                   196             78                        0
## 20-7                   753            206                        0
## 20-8                     0              0                        0
## 21-1                   331             17                       35
## 21-10                  107              0                        0
## 21-11                  200            209                       15
## 21-12                    0              0                      473
## 21-13                   23            114                        0
## 21-14                    0            655                      153
## 21-15                    0              0                       37
## 21-2                     0              0                        0
## 21-3                     0              0                        0
## 21-4                     0              0                        0
## 21-5                     0              0                      796
## 21-6                     0             78                        0
## 21-7                   157              0                      156
## 21-8                   146              0                        0
## 21-9                     0              0                        0
## 28-1                     0              0                       65
## 28-10                  108              0                        0
## 28-11                   38              0                        0
## 28-12                   34              0                        0
## 28-13                   41            441                        0
## 28-14                    0            950                        0
## 28-15                   36            287                      249
## 28-2                    21            100                        0
## 28-3                   678            159                       43
## 28-4                     0             42                      254
## 28-5                     0            201                      242
## 28-6                    52            537                        0
## 28-7                    46            186                       45
## 28-8                   172            972                        0
## 28-9                     0              0                     1085
## 29-1                    75            328                        0
## 29-10                    0              0                     1174
## 29-11                   61             14                       17
## 29-12                  168             16                        0
## 29-13                  574            535                       54
## 29-14                    0            192                        0
## 29-15                    0              0                        0
## 29-2                   193            256                        0
## 29-3                     0            112                      257
## 29-4                   275            242                        0
## 29-5                   184            497                        0
## 29-6                    82            240                        0
## 29-7                   115            199                        0
## 29-8                   138            149                        0
## 29-9                     0              0                        0
##       Quadrisphaera granulorum Aureimonas sp..1 Klenkia sp.
## 20-10                        0                0           0
## 20-11                        0                0           0
## 20-12                        0                0           0
## 20-13                      993                0         892
## 20-14                      112                0          29
## 20-15                        0                0           0
## 20-2                        24                0           0
## 20-3                       158                0           0
## 20-4                       140                0           0
## 20-5                       238              215           0
## 20-6                         0                0          51
## 20-7                         0                0           0
## 20-8                         0                0           0
## 21-1                        10                0          55
## 21-10                       39                0           0
## 21-11                        0                0           0
## 21-12                        0                0           0
## 21-13                        0                0           0
## 21-14                        0                0          26
## 21-15                        0                0           0
## 21-2                         0                0           0
## 21-3                        30               20          33
## 21-4                       257               19          11
## 21-5                        34                0           0
## 21-6                         0               35         190
## 21-7                        51              312           6
## 21-8                        52                0          29
## 21-9                         0                0           0
## 28-1                         0                0           0
## 28-10                        0                0           0
## 28-11                       14                0           0
## 28-12                        0               93         372
## 28-13                        0               41           0
## 28-14                       62               52           0
## 28-15                      215                0           0
## 28-2                         0                0          88
## 28-3                         0                0           0
## 28-4                       131               54           0
## 28-5                       315                0          39
## 28-6                         0               17           0
## 28-7                         0                0          17
## 28-8                         0               36           5
## 28-9                         0                0           0
## 29-1                       131                0           0
## 29-10                        0                0           0
## 29-11                        5              699           0
## 29-12                        0                0           0
## 29-13                      292               33          87
## 29-14                      144               85         128
## 29-15                        0                0           0
## 29-2                         0                0           0
## 29-3                         0              673           0
## 29-4                       166               35           5
## 29-5                        68                0          29
## 29-6                         0                0           0
## 29-7                        33              356         114
## 29-8                       122               62           9
## 29-9                         6                0           0
##       Pseudokineococcus lusitanus Methylobacterium-Methylorubrum sp..2
## 20-10                           0                                    0
## 20-11                           0                                    0
## 20-12                           0                                    0
## 20-13                          56                                    0
## 20-14                          64                                 1011
## 20-15                           0                                    0
## 20-2                            0                                    0
## 20-3                          160                                    0
## 20-4                            0                                    0
## 20-5                           55                                    0
## 20-6                            0                                    0
## 20-7                          193                                    0
## 20-8                            0                                    0
## 21-1                            0                                    0
## 21-10                         380                                    0
## 21-11                          27                                    0
## 21-12                           0                                    0
## 21-13                          45                                    0
## 21-14                           0                                    0
## 21-15                           0                                    0
## 21-2                            0                                    0
## 21-3                           30                                    0
## 21-4                           31                                    0
## 21-5                            0                                    0
## 21-6                          599                                  236
## 21-7                            0                                    0
## 21-8                            0                                    0
## 21-9                            0                                    0
## 28-1                            0                                    0
## 28-10                           0                                    0
## 28-11                           0                                    0
## 28-12                           0                                    0
## 28-13                           0                                    0
## 28-14                           0                                    0
## 28-15                           0                                    0
## 28-2                            0                                    0
## 28-3                            0                                    0
## 28-4                            0                                    0
## 28-5                            0                                    0
## 28-6                           42                                    0
## 28-7                            0                                    0
## 28-8                            0                                    0
## 28-9                            0                                    0
## 29-1                           96                                    0
## 29-10                           0                                    0
## 29-11                           0                                    0
## 29-12                          49                                    0
## 29-13                          15                                  300
## 29-14                          75                                  197
## 29-15                           0                                    0
## 29-2                            0                                    0
## 29-3                          346                                    0
## 29-4                           33                                    0
## 29-5                            0                                    0
## 29-6                           58                                    0
## 29-7                            0                                    0
## 29-8                           32                                    0
## 29-9                           32                                    0
##       Aureimonas populi Sphingomonas sp. Methylobacterium-Methylorubrum sp..3
## 20-10                 0                0                                    0
## 20-11                 0                0                                  185
## 20-12                 0                4                                   16
## 20-13                42                0                                    0
## 20-14                 0                0                                    0
## 20-15                 0                0                                    0
## 20-2                  0                0                                    0
## 20-3                  0                0                                    0
## 20-4                  0                0                                    0
## 20-5                108              259                                  519
## 20-6                  0                0                                    0
## 20-7                  0                0                                    0
## 20-8                  0                0                                    0
## 21-1                  0                0                                   65
## 21-10               164              338                                    0
## 21-11               122               26                                    0
## 21-12                 0                0                                    0
## 21-13                 0               26                                   16
## 21-14                 0                0                                    0
## 21-15                 0                0                                    0
## 21-2                  0                0                                    0
## 21-3                  0              329                                    0
## 21-4                  0                0                                    0
## 21-5                  0                0                                    0
## 21-6                  0                0                                    0
## 21-7                123                0                                    0
## 21-8                  0                0                                    0
## 21-9                  0                0                                    0
## 28-1                  0               39                                    0
## 28-10                 0               68                                    0
## 28-11                 0               16                                    0
## 28-12                 0                0                                    0
## 28-13                 0                0                                   19
## 28-14                 0                0                                    0
## 28-15                50               81                                    0
## 28-2                  0                0                                    0
## 28-3                  0                0                                    0
## 28-4                112               92                                    0
## 28-5                  0               48                                    0
## 28-6                133                0                                    0
## 28-7                 21                0                                    0
## 28-8                163               51                                    0
## 28-9                  0               56                                    0
## 29-1                 18               36                                   29
## 29-10                 0                0                                    0
## 29-11                29              225                                    0
## 29-12                 0                0                                    0
## 29-13                 0                0                                    0
## 29-14               398              165                                    0
## 29-15                 0                0                                    0
## 29-2                  0               34                                   88
## 29-3                  0               94                                    0
## 29-4                  0               74                                  161
## 29-5                  0               36                                    0
## 29-6                  0                0                                    0
## 29-7                  0               14                                    0
## 29-8                  0               94                                    0
## 29-9                  0                0                                    0
##       Methylobacterium-Methylorubrum sp..4 Aurantimonas sp.
## 20-10                                    0                0
## 20-11                                    0                0
## 20-12                                    0                0
## 20-13                                    0                0
## 20-14                                    0                0
## 20-15                                    0                0
## 20-2                                     0                0
## 20-3                                     0              213
## 20-4                                     0                0
## 20-5                                     0               54
## 20-6                                     0                0
## 20-7                                     0              101
## 20-8                                     0                0
## 21-1                                     0                0
## 21-10                                    0                0
## 21-11                                    0                0
## 21-12                                    0                0
## 21-13                                    0                0
## 21-14                                    0                0
## 21-15                                    0                0
## 21-2                                     0                0
## 21-3                                     0                9
## 21-4                                     0                0
## 21-5                                     0                0
## 21-6                                     0                0
## 21-7                                     0                0
## 21-8                                     0               32
## 21-9                                     0                0
## 28-1                                     0                0
## 28-10                                    0                0
## 28-11                                    0                0
## 28-12                                    0                0
## 28-13                                    0                0
## 28-14                                    0                0
## 28-15                                    0                0
## 28-2                                     0                0
## 28-3                                     0                0
## 28-4                                     0                0
## 28-5                                     0                0
## 28-6                                     0                0
## 28-7                                     0                0
## 28-8                                     0                0
## 28-9                                     0                0
## 29-1                                     0               69
## 29-10                                    0              210
## 29-11                                    0                0
## 29-12                                    0                0
## 29-13                                    0              150
## 29-14                                    0               56
## 29-15                                    0                0
## 29-2                                     0                0
## 29-3                                     0                0
## 29-4                                     0               47
## 29-5                                     0                0
## 29-6                                     0                0
## 29-7                                     0                0
## 29-8                                     0                0
## 29-9                                     0               18
##       Methylobacterium-Methylorubrum sp..5 Methylobacterium-Methylorubrum sp..6
## 20-10                                    0                                    0
## 20-11                                    0                                    0
## 20-12                                    0                                    0
## 20-13                                    0                                    0
## 20-14                                    0                                    0
## 20-15                                    0                                    0
## 20-2                                     0                                    0
## 20-3                                     0                                    0
## 20-4                                     0                                    0
## 20-5                                     0                                    0
## 20-6                                     0                                    0
## 20-7                                     0                                    0
## 20-8                                     0                                    0
## 21-1                                     0                                    0
## 21-10                                    0                                    0
## 21-11                                    0                                    0
## 21-12                                    0                                    0
## 21-13                                    0                                    0
## 21-14                                    0                                    0
## 21-15                                    0                                    0
## 21-2                                     0                                    0
## 21-3                                     0                                    0
## 21-4                                     0                                    0
## 21-5                                     0                                    0
## 21-6                                     0                                    0
## 21-7                                     0                                    0
## 21-8                                     0                                    0
## 21-9                                     0                                    0
## 28-1                                     0                                    0
## 28-10                                    0                                    0
## 28-11                                    0                                    0
## 28-12                                    0                                    0
## 28-13                                    0                                    0
## 28-14                                    0                                    0
## 28-15                                    0                                  923
## 28-2                                     0                                    0
## 28-3                                     0                                    0
## 28-4                                     0                                    0
## 28-5                                     0                                    0
## 28-6                                     0                                    0
## 28-7                                     0                                    0
## 28-8                                     0                                    0
## 28-9                                     0                                    0
## 29-1                                     0                                    0
## 29-10                                    0                                    0
## 29-11                                    0                                    0
## 29-12                                    0                                    0
## 29-13                                    0                                    0
## 29-14                                    0                                    0
## 29-15                                    0                                    0
## 29-2                                     0                                    0
## 29-3                                     0                                    0
## 29-4                                     0                                    0
## 29-5                                     0                                    0
## 29-6                                     0                                    0
## 29-7                                     0                                    0
## 29-8                                     0                                    0
## 29-9                                     0                                    0
##       Methylobacterium-Methylorubrum sp..7 Sphingomonas mucosissima
## 20-10                                    0                        0
## 20-11                                    0                        0
## 20-12                                    0                        0
## 20-13                                    0                        0
## 20-14                                    0                        0
## 20-15                                    0                        0
## 20-2                                     0                        0
## 20-3                                     0                        0
## 20-4                                     0                        0
## 20-5                                     0                        0
## 20-6                                     0                        0
## 20-7                                     0                        0
## 20-8                                     0                        0
## 21-1                                     0                        0
## 21-10                                    0                        0
## 21-11                                    0                        0
## 21-12                                    0                        0
## 21-13                                    0                        0
## 21-14                                    0                       47
## 21-15                                    0                        0
## 21-2                                     0                        0
## 21-3                                     0                        0
## 21-4                                     0                        0
## 21-5                                     0                        0
## 21-6                                     0                        0
## 21-7                                     0                        0
## 21-8                                     0                        0
## 21-9                                     0                        0
## 28-1                                     0                        0
## 28-10                                    0                        0
## 28-11                                    0                        0
## 28-12                                    0                        0
## 28-13                                    0                        0
## 28-14                                    0                      341
## 28-15                                    0                        0
## 28-2                                     0                        0
## 28-3                                     0                        0
## 28-4                                     0                        0
## 28-5                                     0                        0
## 28-6                                    24                        0
## 28-7                                     0                        0
## 28-8                                     0                        0
## 28-9                                     0                        0
## 29-1                                     0                        0
## 29-10                                    0                        0
## 29-11                                    0                        0
## 29-12                                    0                        0
## 29-13                                    0                      394
## 29-14                                    0                        0
## 29-15                                    0                        0
## 29-2                                     0                        0
## 29-3                                     0                        0
## 29-4                                     0                        0
## 29-5                                    59                        0
## 29-6                                     0                        0
## 29-7                                     0                        0
## 29-8                                     0                        0
## 29-9                                     0                        0
##       Bdellovibrio sp. Methylobacterium-Methylorubrum sp..8 Quadrisphaera sp.
## 20-10                0                                    0                 0
## 20-11                0                                    0                 0
## 20-12                0                                    0                 0
## 20-13                0                                    0                 0
## 20-14                0                                    0                 0
## 20-15                0                                    0                 0
## 20-2                 0                                    0                 0
## 20-3                 0                                    0                 0
## 20-4                 0                                    0                 0
## 20-5                 0                                    0                 0
## 20-6                 0                                    0                 0
## 20-7                 0                                    0                 0
## 20-8                 0                                    0                 0
## 21-1                 0                                    0                 0
## 21-10                0                                    0                 0
## 21-11               36                                    0                 0
## 21-12                0                                    0                 0
## 21-13                0                                    0                 0
## 21-14                0                                    0                 0
## 21-15                0                                    0                 0
## 21-2                 0                                    0                 0
## 21-3                 0                                    0                 0
## 21-4               389                                    0                 0
## 21-5                 0                                    0                 0
## 21-6                 0                                    0                 0
## 21-7                 0                                    0                 0
## 21-8                 0                                    0                 0
## 21-9                 0                                    0                 0
## 28-1                 0                                    0                 0
## 28-10                0                                    0                 0
## 28-11                0                                    0                 0
## 28-12                0                                    0                 0
## 28-13                0                                    0                 0
## 28-14                0                                    0                 0
## 28-15                0                                    0                 0
## 28-2                 0                                    0                 0
## 28-3                 0                                    0                 0
## 28-4                 0                                    0                 0
## 28-5                 0                                    0                 0
## 28-6                 0                                    0                 0
## 28-7                 0                                    0                 0
## 28-8                 0                                    0                 0
## 28-9                 0                                    0                 0
## 29-1                 0                                    0                 0
## 29-10                0                                    0                 0
## 29-11                0                                    0                 0
## 29-12                0                                    0                 0
## 29-13                0                                    0                 0
## 29-14                0                                    0                 0
## 29-15                0                                    0                 0
## 29-2                 0                                    0                 0
## 29-3                 0                                    0                 0
## 29-4                 0                                    0                 0
## 29-5                 0                                    0                 0
## 29-6                 0                                    0                 0
## 29-7                 0                                    0                 0
## 29-8                22                                    0                 0
## 29-9                 0                                    0                 0
##       Hymenobacter rivuli Methylobacterium-Methylorubrum sp..9
## 20-10                   0                                    0
## 20-11                   0                                    0
## 20-12                   0                                    0
## 20-13                   0                                    0
## 20-14                   0                                    0
## 20-15                   0                                    0
## 20-2                    0                                    0
## 20-3                    0                                    0
## 20-4                    0                                    0
## 20-5                    0                                    0
## 20-6                    0                                    0
## 20-7                    0                                    0
## 20-8                    0                                    0
## 21-1                    0                                    0
## 21-10                   0                                    0
## 21-11                   0                                    0
## 21-12                   0                                    0
## 21-13                   0                                    0
## 21-14                   0                                    0
## 21-15                   0                                    0
## 21-2                    0                                    0
## 21-3                    0                                    0
## 21-4                    0                                    0
## 21-5                    0                                    0
## 21-6                    0                                    0
## 21-7                    0                                    0
## 21-8                    0                                    0
## 21-9                    0                                    0
## 28-1                    0                                    0
## 28-10                   0                                    0
## 28-11                   0                                    0
## 28-12                   0                                    0
## 28-13                   0                                    0
## 28-14                   0                                    0
## 28-15                   0                                    0
## 28-2                    0                                    0
## 28-3                    0                                    0
## 28-4                    0                                    0
## 28-5                    0                                    0
## 28-6                    0                                  545
## 28-7                    0                                    0
## 28-8                    0                                    0
## 28-9                    0                                    0
## 29-1                    0                                    0
## 29-10                   0                                    0
## 29-11                   0                                    0
## 29-12                   0                                    0
## 29-13                   0                                    0
## 29-14                   0                                    0
## 29-15                   0                                    0
## 29-2                    0                                    0
## 29-3                    0                                    0
## 29-4                    0                                    0
## 29-5                    0                                    0
## 29-6                    0                                    0
## 29-7                  281                                    0
## 29-8                    0                                    0
## 29-9                    0                                    0
##       Aurantimonas sp..1 Methylobacterium-Methylorubrum sp..10
## 20-10                  0                                     0
## 20-11                  0                                     0
## 20-12                  0                                     0
## 20-13                  0                                     0
## 20-14                  0                                     0
## 20-15                  0                                     0
## 20-2                   0                                     0
## 20-3                   0                                     0
## 20-4                   0                                     0
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## 29-8                   0                                     0
## 29-9                   0                                     0
##       Sphingomonas sp..1 Methylobacterium-Methylorubrum sp..11 Hymenobacter sp.
## 20-10                  0                                     0                0
## 20-11                  0                                     0                0
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## 29-7                   0                                     0              235
## 29-8                   0                                     0                0
## 29-9                   0                                     0                0
##       Escherichia-Shigella sp..1 Aureimonas sp..2
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## 29-9                           0                0
##       Methylobacterium-Methylorubrum sp..12 Herbiconiux sp. Oscillatoria sp.
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## 29-9                                      0               0                0
##       Microbacterium sp. Methylobacterium-Methylorubrum sp..13
## 20-10                  0                                     0
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## 20-14                  0                                     0
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## 29-10                 51                                     0
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## 29-6                   0                                     0
## 29-7                   0                                     0
## 29-8                   0                                     0
## 29-9                   0                                     0
##       Hymenobacter sp..1 Aureimonas sp..3 Sphingomonas sp..2
## 20-10                  0                0                  0
## 20-11                  0                0                  0
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## 21-4                   0                0                 70
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## 21-9                   0                0                  0
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## 29-9                   0                0                  0
##       Methylobacterium-Methylorubrum sp..14
## 20-10                                     0
## 20-11                                     0
## 20-12                                     0
## 20-13                                     0
## 20-14                                     0
## 20-15                                     0
## 20-2                                      0
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## 21-7                                      0
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## 28-5                                      0
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## 28-7                                      0
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## 28-9                                      0
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## 29-13                                   199
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## 29-6                                      0
## 29-7                                      0
## 29-8                                      0
## 29-9                                      0
##       Methylobacterium-Methylorubrum sp..15 Aureimonas sp..4 Hymenobacter sp..2
## 20-10                                     0                0                  0
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## 21-9                                      0                0                  0
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## 28-15                                     0                0                 10
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## 29-6                                      0                0                  0
## 29-7                                      0                0                  0
## 29-8                                      0                0                  0
## 29-9                                      0                0                  0
##       Ensifer sp. Methylobacterium-Methylorubrum sp..16 Oscillatoria sp..1
## 20-10           0                                     0                  0
## 20-11           0                                     0                  0
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## 29-7            0                                     0                  0
## 29-8            0                                     0                  0
## 29-9            0                                     0                  0
##       Marmoricola sp. Methylobacterium-Methylorubrum sp..17
## 20-10               0                                     0
## 20-11               0                                     0
## 20-12               0                                     0
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## 28-5                0                                     0
## 28-6                0                                     0
## 28-7                0                                     0
## 28-8                0                                     0
## 28-9                0                                     0
## 29-1                0                                     0
## 29-10               0                                     0
## 29-11               0                                     0
## 29-12               0                                     0
## 29-13               0                                     0
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## 29-5                0                                     0
## 29-6                0                                     0
## 29-7                0                                     0
## 29-8                0                                     0
## 29-9                0                                     0
##       Methylobacterium-Methylorubrum sp..18 Hymenobacter sp..3
## 20-10                                     0                  0
## 20-11                                     0                  0
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## 21-13                                     0                162
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## 21-4                                      0                  0
## 21-5                                    166                  0
## 21-6                                      0                  0
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## 28-5                                      0                  0
## 28-6                                      0                  0
## 28-7                                      0                  0
## 28-8                                      0                  0
## 28-9                                      0                  0
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## 29-5                                      0                  0
## 29-6                                      0                  0
## 29-7                                      0                  0
## 29-8                                      0                  0
## 29-9                                      0                  0
##       Sphingomonas sp..3 Oscillatoria sp..2
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## 20-11                  0                  0
## 20-12                  0                  0
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##       Methylobacterium-Methylorubrum sp..19
## 20-10                                     0
## 20-11                                     0
## 20-12                                     0
## 20-13                                     0
## 20-14                                     0
## 20-15                                     0
## 20-2                                      0
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## 21-9                                      0
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## 28-12                                     0
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## 28-15                                     0
## 28-2                                      0
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## 28-5                                      0
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## 28-7                                      0
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## 29-5                                      0
## 29-6                                      0
## 29-7                                      0
## 29-8                                      0
## 29-9                                      0
##       Methylobacterium-Methylorubrum sp..20 Oscillatoria sp..3
## 20-10                                     0                  0
## 20-11                                     0                  0
## 20-12                                     0                  0
## 20-13                                     0                  0
## 20-14                                     0                  0
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## 29-5                                      0                  0
## 29-6                                      0                  0
## 29-7                                      0                  0
## 29-8                                      0                  0
## 29-9                                      0                  0
##       Sphingomonas sp..4 Oscillatoria sp..4
## 20-10                  0                141
## 20-11                  0                  0
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## 29-1                   0                  0
## 29-10                  0                  0
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## 29-12                  0                  0
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## 29-15                  0                  0
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## 29-6                   0                  0
## 29-7                   0                  0
## 29-8                   0                  0
## 29-9                   0                  0
##       Methylobacterium-Methylorubrum sp..21 Thermus sp.
## 20-10                                     0           0
## 20-11                                     0           0
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## 29-8                                      0           0
## 29-9                                      0           0
##       Methylobacterium-Methylorubrum sp..22
## 20-10                                     0
## 20-11                                     0
## 20-12                                     0
## 20-13                                     0
## 20-14                                     0
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## 20-2                                      0
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## 29-8                                      0
## 29-9                                      0
##       Methylobacterium-Methylorubrum sp..23 Quadrisphaera sp..1
## 20-10                                     0                   0
## 20-11                                     0                   0
## 20-12                                     0                   0
## 20-13                                     0                   0
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## 29-8                                      0                   0
## 29-9                                      0                   0
##       Methylobacterium-Methylorubrum sp..24 Acinetobacter sp.
## 20-10                                     0                 0
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## 20-8                                      0               130
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## 29-7                                      0                 0
## 29-8                                      0                 0
## 29-9                                      0                 0
##       Methylobacterium-Methylorubrum sp..25
## 20-10                                     0
## 20-11                                     0
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## 28-1                                      0
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## 28-12                                     0
## 28-13                                     0
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## 28-9                                      0
## 29-1                                      0
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## 29-13                                     0
## 29-14                                   128
## 29-15                                     0
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## 29-5                                      0
## 29-6                                      0
## 29-7                                      0
## 29-8                                      0
## 29-9                                      0
##       Methylobacterium-Methylorubrum sp..26 Oscillatoria sp..5
## 20-10                                     0                  0
## 20-11                                     0                  0
## 20-12                                     0                  0
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## 29-5                                      0                  0
## 29-6                                      0                  0
## 29-7                                      0                  0
## 29-8                                      0                  0
## 29-9                                      0                  0
##       Methylobacterium-Methylorubrum sp..27
## 20-10                                     0
## 20-11                                     0
## 20-12                                     0
## 20-13                                     0
## 20-14                                     0
## 20-15                                     0
## 20-2                                      0
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## 21-4                                      0
## 21-5                                      0
## 21-6                                      0
## 21-7                                      0
## 21-8                                      0
## 21-9                                      0
## 28-1                                      0
## 28-10                                     0
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## 28-13                                     0
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## 28-2                                      0
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## 28-5                                      0
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## 28-9                                      0
## 29-1                                      0
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## 29-5                                      0
## 29-6                                      0
## 29-7                                      0
## 29-8                                      0
## 29-9                                      0
##       Methylobacterium-Methylorubrum sp..28 Oscillatoria sp..6
## 20-10                                     0                  0
## 20-11                                     0                  0
## 20-12                                     0                  0
## 20-13                                     0                  0
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## 21-6                                      0                109
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## 28-4                                      0                  0
## 28-5                                      0                  0
## 28-6                                      0                  0
## 28-7                                      0                  0
## 28-8                                      0                  0
## 28-9                                      0                  0
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## 29-12                                     0                  0
## 29-13                                     0                  0
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## 29-5                                      0                  0
## 29-6                                      0                  0
## 29-7                                      0                  0
## 29-8                                      0                  0
## 29-9                                      0                  0
##       Sphingomonas sp..5 Oscillatoria sp..7 Pseudomonas sp.
## 20-10                  0                  0               0
## 20-11                  0                  0               0
## 20-12                  0                  0               0
## 20-13                  0                  0               0
## 20-14                  0                  0               0
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## 21-15                  0                107               0
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## 21-9                   0                  0               0
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## 28-4                   0                  0              91
## 28-5                   0                  0               0
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## 29-1                   0                  0               0
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## 29-7                   0                  0               0
## 29-8                   0                  0               0
## 29-9                   0                  0               0
##       Pseudokineococcus sp. Oscillatoria sp..8 Ensifer sp..1
## 20-10                     0                  0             0
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##       Escherichia-Shigella sp..2 Methylobacterium-Methylorubrum sp..29
## 20-10                          0                                     0
## 20-11                          0                                     0
## 20-12                          0                                     0
## 20-13                          0                                     0
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## 28-5                           0                                     0
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## 28-8                           0                                     0
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## 29-6                           0                                     0
## 29-7                           0                                     0
## 29-8                           0                                     0
## 29-9                           0                                     0
##       Oscillatoria sp..9 Methylobacterium-Methylorubrum sp..30
## 20-10                  0                                     0
## 20-11                  0                                     0
## 20-12                  0                                     0
## 20-13                  0                                     0
## 20-14                  0                                     0
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## 21-12                  0                                     0
## 21-13                  0                                     0
## 21-14                  0                                     0
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## 21-3                   0                                     0
## 21-4                   0                                     0
## 21-5                   0                                     0
## 21-6                   0                                     0
## 21-7                   0                                     0
## 21-8                   0                                     0
## 21-9                   0                                     0
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## 28-10                  0                                     0
## 28-11                  0                                     0
## 28-12                  0                                     0
## 28-13                  0                                     0
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## 28-15                  0                                     0
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## 28-3                   0                                     0
## 28-4                   0                                     0
## 28-5                   0                                     0
## 28-6                  96                                     0
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## 28-8                   0                                     0
## 28-9                   0                                     0
## 29-1                   0                                     0
## 29-10                  0                                     0
## 29-11                  0                                     0
## 29-12                  0                                     0
## 29-13                  0                                     0
## 29-14                  0                                     0
## 29-15                  0                                     0
## 29-2                   0                                    96
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## 29-4                   0                                     0
## 29-5                   0                                     0
## 29-6                   0                                     0
## 29-7                   0                                     0
## 29-8                   0                                     0
## 29-9                   0                                     0
##       Novosphingobium sp. Methylobacterium-Methylorubrum sp..31
## 20-10                   0                                     0
## 20-11                   0                                     0
## 20-12                   0                                     0
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## 20-14                   0                                     0
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## 28-13                   0                                     0
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## 29-11                   0                                     0
## 29-12                   0                                     0
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## 29-3                    0                                     0
## 29-4                    0                                     0
## 29-5                    0                                     0
## 29-6                    0                                     0
## 29-7                    0                                     0
## 29-8                    0                                     0
## 29-9                    0                                     0
##       Escherichia-Shigella sp..3 Oscillatoria sp..10
## 20-10                          0                   0
## 20-11                          0                   0
## 20-12                          0                   0
## 20-13                          0                  85
## 20-14                          0                   0
## 20-15                          0                   0
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## 21-10                          0                   0
## 21-11                          0                   0
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## 21-13                          0                   0
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## 21-2                           0                   0
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## 21-4                           0                   0
## 21-5                           0                   0
## 21-6                           0                   0
## 21-7                           0                   0
## 21-8                           0                   0
## 21-9                           0                   0
## 28-1                           0                   0
## 28-10                          0                   0
## 28-11                          0                   0
## 28-12                          0                   0
## 28-13                          0                   0
## 28-14                          0                   0
## 28-15                          0                   0
## 28-2                           0                   0
## 28-3                           0                   0
## 28-4                           0                   0
## 28-5                           0                   0
## 28-6                           0                   0
## 28-7                           0                   0
## 28-8                           0                   0
## 28-9                           0                   0
## 29-1                           0                   0
## 29-10                          0                   0
## 29-11                          0                   0
## 29-12                          0                   0
## 29-13                          0                   0
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## 29-5                           0                   0
## 29-6                           0                   0
## 29-7                           0                   0
## 29-8                           0                   0
## 29-9                           0                   0
##       Methylobacterium-Methylorubrum sp..32 Neorhizobium sp. Sphingomonas sp..6
## 20-10                                     0                0                  0
## 20-11                                     0                0                  0
## 20-12                                     0                0                  0
## 20-13                                     0                0                  0
## 20-14                                     0                0                  0
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## 21-2                                      0                0                  0
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## 21-4                                      0                0                  0
## 21-5                                      0                0                  0
## 21-6                                      0                0                 80
## 21-7                                      0                0                  0
## 21-8                                      0                0                  0
## 21-9                                      0                0                  0
## 28-1                                      0                0                  0
## 28-10                                     0                0                  0
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## 28-12                                     0                0                  0
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## 28-2                                      0                0                  0
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## 28-5                                      0                0                  0
## 28-6                                      0                0                  0
## 28-7                                      0                0                  0
## 28-8                                      0                0                  0
## 28-9                                      0                0                  0
## 29-1                                      0                0                  0
## 29-10                                     0                0                  0
## 29-11                                     0                0                  0
## 29-12                                     0                0                  0
## 29-13                                     0                0                  0
## 29-14                                     0                0                  0
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## 29-2                                      0                0                  0
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## 29-4                                      0                0                  0
## 29-5                                      0                0                  0
## 29-6                                      0                0                  0
## 29-7                                      0                0                  0
## 29-8                                      0                0                  0
## 29-9                                      0                0                  0
##       Hymenobacter sp..4 Methylobacterium-Methylorubrum sp..33
## 20-10                  0                                     0
## 20-11                  0                                     0
## 20-12                  0                                     0
## 20-13                  9                                     0
## 20-14                  0                                     0
## 20-15                  0                                     0
## 20-2                   0                                     0
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## 20-4                   0                                     0
## 20-5                   0                                     0
## 20-6                   0                                     0
## 20-7                   0                                     0
## 20-8                   0                                     0
## 21-1                   0                                     0
## 21-10                  0                                     0
## 21-11                  0                                     0
## 21-12                  0                                     0
## 21-13                  0                                     0
## 21-14                  0                                     0
## 21-15                  0                                     0
## 21-2                   0                                     0
## 21-3                   0                                     0
## 21-4                   0                                     0
## 21-5                   0                                     0
## 21-6                   0                                     0
## 21-7                   0                                     0
## 21-8                   0                                     0
## 21-9                   0                                     0
## 28-1                   0                                     0
## 28-10                  0                                     0
## 28-11                  0                                     0
## 28-12                  0                                     0
## 28-13                  0                                     0
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## 28-15                  0                                     0
## 28-2                   0                                     0
## 28-3                   0                                     0
## 28-4                   0                                     0
## 28-5                   0                                     0
## 28-6                   0                                     0
## 28-7                   0                                     0
## 28-8                   0                                     0
## 28-9                   0                                     0
## 29-1                   0                                     0
## 29-10                  0                                     0
## 29-11                  0                                     0
## 29-12                  0                                     0
## 29-13                  0                                     0
## 29-14                  0                                     0
## 29-15                  0                                     0
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## 29-3                   0                                     0
## 29-4                   0                                     0
## 29-5                   0                                     0
## 29-6                   0                                     0
## 29-7                  16                                     0
## 29-8                   0                                     0
## 29-9                   0                                     0
##       Oscillatoria sp..11 Methylobacterium-Methylorubrum sp..34
## 20-10                   0                                     0
## 20-11                   0                                     0
## 20-12                   0                                     0
## 20-13                   0                                     0
## 20-14                   0                                     0
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## 21-1                    0                                     0
## 21-10                   0                                     0
## 21-11                   0                                     0
## 21-12                   0                                     0
## 21-13                   0                                     0
## 21-14                   0                                     0
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## 21-3                    0                                     0
## 21-4                    0                                     0
## 21-5                    0                                     0
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## 28-1                    0                                     0
## 28-10                   0                                     0
## 28-11                   0                                     0
## 28-12                   0                                     0
## 28-13                   0                                     0
## 28-14                   0                                     0
## 28-15                   0                                     0
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## 28-3                    0                                     0
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## 28-5                    0                                     0
## 28-6                    0                                     0
## 28-7                    0                                     0
## 28-8                    0                                     0
## 28-9                    0                                     0
## 29-1                    0                                     0
## 29-10                   0                                     0
## 29-11                   0                                     0
## 29-12                   0                                     0
## 29-13                   0                                     0
## 29-14                   0                                     0
## 29-15                   0                                     0
## 29-2                    0                                     0
## 29-3                    0                                     0
## 29-4                    0                                    76
## 29-5                    0                                     0
## 29-6                    0                                     0
## 29-7                    0                                     0
## 29-8                    0                                     0
## 29-9                    0                                     0
##       Sphingomonas sp..7 Methylobacterium-Methylorubrum sp..35
## 20-10                  0                                     0
## 20-11                  0                                     0
## 20-12                  0                                     0
## 20-13                  0                                     0
## 20-14                  0                                     0
## 20-15                  0                                     0
## 20-2                   0                                     0
## 20-3                   0                                     0
## 20-4                   0                                     0
## 20-5                   0                                     0
## 20-6                   0                                     0
## 20-7                   0                                     0
## 20-8                   0                                     0
## 21-1                   0                                     0
## 21-10                  0                                     0
## 21-11                  0                                     0
## 21-12                  0                                     0
## 21-13                  0                                     0
## 21-14                 75                                     0
## 21-15                  0                                     0
## 21-2                   0                                     0
## 21-3                   0                                     0
## 21-4                   0                                     0
## 21-5                   0                                     0
## 21-6                   0                                     0
## 21-7                   0                                     0
## 21-8                   0                                     0
## 21-9                   0                                     0
## 28-1                   0                                     0
## 28-10                  0                                     0
## 28-11                  0                                     0
## 28-12                  0                                     0
## 28-13                  0                                     0
## 28-14                  0                                     0
## 28-15                  0                                     0
## 28-2                   0                                     0
## 28-3                   0                                     0
## 28-4                   0                                     0
## 28-5                   0                                     0
## 28-6                   0                                     0
## 28-7                   0                                     0
## 28-8                   0                                     0
## 28-9                   0                                     0
## 29-1                   0                                     0
## 29-10                  0                                     0
## 29-11                  0                                     0
## 29-12                  0                                     0
## 29-13                  0                                     0
## 29-14                  0                                     0
## 29-15                  0                                     0
## 29-2                   0                                     0
## 29-3                   0                                     0
## 29-4                   0                                     0
## 29-5                   0                                     0
## 29-6                   0                                     0
## 29-7                   0                                     0
## 29-8                   0                                     0
## 29-9                   0                                     0
##       Hymenobacter sp..5 Rathayibacter sp.
## 20-10                  0                 0
## 20-11                  0                 0
## 20-12                  0                 0
## 20-13                  0                 0
## 20-14                  0                 0
## 20-15                  0                 0
## 20-2                   0                 0
## 20-3                   0                 0
## 20-4                   0                 0
## 20-5                   0                 0
## 20-6                   0                 0
## 20-7                   0                 0
## 20-8                   0                 0
## 21-1                   0                 0
## 21-10                  0                 0
## 21-11                  0                 0
## 21-12                  0                 0
## 21-13                  0                13
## 21-14                  0                 0
## 21-15                  0                 0
## 21-2                   0                 0
## 21-3                   0                 0
## 21-4                   0                 0
## 21-5                   0                 0
## 21-6                   0                 0
## 21-7                   0                 0
## 21-8                   0                 0
## 21-9                   0                 0
## 28-1                   0                 0
## 28-10                  0                 0
## 28-11                  0                 0
## 28-12                  0                 0
## 28-13                  0                 0
## 28-14                  0                59
## 28-15                  0                 0
## 28-2                   0                 0
## 28-3                   0                 0
## 28-4                   0                 0
## 28-5                   0                 0
## 28-6                   0                 0
## 28-7                   0                 0
## 28-8                   0                 0
## 28-9                   0                 0
## 29-1                   0                 0
## 29-10                  0                 0
## 29-11                  0                 0
## 29-12                  0                 0
## 29-13                  0                 0
## 29-14                  0                 0
## 29-15                  0                 0
## 29-2                   0                 0
## 29-3                   0                 0
## 29-4                  74                 0
## 29-5                   0                 0
## 29-6                   0                 0
## 29-7                   0                 0
## 29-8                   0                 0
## 29-9                   0                 0
##       Methylobacterium-Methylorubrum sp..36 Oscillatoria sp..12 Erwiniaceae
## 20-10                                     0                   0           0
## 20-11                                     0                   0           0
## 20-12                                     0                   0           0
## 20-13                                     0                   0           0
## 20-14                                     0                   0           0
## 20-15                                     0                   0           0
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## 20-6                                      0                   0           0
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## 20-8                                      0                   0           0
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## 21-10                                     0                   0           0
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## 21-3                                      0                   0           0
## 21-4                                     71                   0           0
## 21-5                                      0                   0           0
## 21-6                                      0                   0           0
## 21-7                                      0                   0           0
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## 21-9                                      0                   0           0
## 28-1                                      0                   0           0
## 28-10                                     0                   0           0
## 28-11                                     0                   0           0
## 28-12                                     0                   0           0
## 28-13                                     0                   0           0
## 28-14                                     0                   0           0
## 28-15                                     0                   0           0
## 28-2                                      0                   0           0
## 28-3                                      0                   0           0
## 28-4                                      0                   0           0
## 28-5                                      0                   0           0
## 28-6                                      0                   0           0
## 28-7                                      0                   0           0
## 28-8                                      0                   0           0
## 28-9                                      0                   0           0
## 29-1                                      0                   0           0
## 29-10                                     0                   0           0
## 29-11                                     0                   0           0
## 29-12                                     0                   0           0
## 29-13                                     0                   0          67
## 29-14                                     0                   0           0
## 29-15                                     0                   0           0
## 29-2                                      0                   0           0
## 29-3                                      0                   0           0
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## 29-5                                      0                   0           0
## 29-6                                      0                   0           0
## 29-7                                      0                   0           0
## 29-8                                      0                   0           0
## 29-9                                      0                   0           0
##       Aureimonas sp..5 Oscillatoria sp..13
## 20-10                0                   0
## 20-11                0                   0
## 20-12                0                   0
## 20-13                0                   0
## 20-14                0                   0
## 20-15                0                   0
## 20-2                 0                   0
## 20-3                 0                   0
## 20-4                 0                   0
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## 20-6                 0                   0
## 20-7                 0                   0
## 20-8                 0                   0
## 21-1                 0                   0
## 21-10                0                   0
## 21-11                0                   0
## 21-12                0                   0
## 21-13                0                   0
## 21-14                0                   0
## 21-15                0                   0
## 21-2                 0                   0
## 21-3                 0                   0
## 21-4                 0                   0
## 21-5                 0                   0
## 21-6                 0                   0
## 21-7                 0                   0
## 21-8                 0                   0
## 21-9                 0                   0
## 28-1                 0                   0
## 28-10                0                   0
## 28-11                0                   0
## 28-12                0                   0
## 28-13                0                   0
## 28-14                0                   0
## 28-15                0                   0
## 28-2                 0                   0
## 28-3                66                   0
## 28-4                 0                   0
## 28-5                 0                   0
## 28-6                 0                   0
## 28-7                 0                   0
## 28-8                 0                   0
## 28-9                 0                   0
## 29-1                 0                   0
## 29-10                0                   0
## 29-11                0                   0
## 29-12                0                   0
## 29-13                0                   0
## 29-14                0                   0
## 29-15                0                   0
## 29-2                 0                   0
## 29-3                 0                   0
## 29-4                 0                   0
## 29-5                 0                   0
## 29-6                 0                   0
## 29-7                 0                   0
## 29-8                 0                   0
## 29-9                 0                   0
##       Methylobacterium-Methylorubrum sp..37 Roseomonas sp. Quadrisphaera sp..2
## 20-10                                     0              0                   0
## 20-11                                     0              0                   0
## 20-12                                     0              0                   0
## 20-13                                     0              0                   0
## 20-14                                     0              0                   0
## 20-15                                     0              0                   0
## 20-2                                      0              0                   0
## 20-3                                      0              0                   0
## 20-4                                     64              0                   0
## 20-5                                      0              0                   0
## 20-6                                      0              0                   0
## 20-7                                      0              0                   0
## 20-8                                      0              0                   0
## 21-1                                      0              0                   0
## 21-10                                     0              0                   0
## 21-11                                     0              0                   0
## 21-12                                     0              0                   0
## 21-13                                     0              0                   0
## 21-14                                     0              0                   0
## 21-15                                     0              0                   0
## 21-2                                      0              0                   0
## 21-3                                      0              0                   0
## 21-4                                      0              0                   0
## 21-5                                      0              0                   0
## 21-6                                      0             32                   0
## 21-7                                      0              0                   0
## 21-8                                      0              0                   0
## 21-9                                      0              0                   0
## 28-1                                      0              0                   0
## 28-10                                     0              0                   0
## 28-11                                     0              0                   0
## 28-12                                     0              0                   0
## 28-13                                     0              0                   0
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## 28-15                                     0              0                   0
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## 28-5                                      0              0                   0
## 28-6                                      0              0                   0
## 28-7                                      0              0                   0
## 28-8                                      0              0                   0
## 28-9                                      0              0                   0
## 29-1                                      0              0                   0
## 29-10                                     0              0                   0
## 29-11                                     0              0                   0
## 29-12                                     0              0                   0
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## 29-14                                     0              0                   0
## 29-15                                     0              0                   0
## 29-2                                      0              0                   0
## 29-3                                      0              0                   0
## 29-4                                      0              0                   0
## 29-5                                      0              0                   0
## 29-6                                      0              0                   0
## 29-7                                      0              0                   0
## 29-8                                      0              0                   0
## 29-9                                      0              0                   0
##       Roseomonas sp..1 Sphingomonas sp..8 Oscillatoria sp..14
## 20-10                0                  0                   0
## 20-11                0                  0                   0
## 20-12                0                  0                   0
## 20-13                0                  0                   0
## 20-14                0                  0                   0
## 20-15                0                  0                   0
## 20-2                 0                  0                   0
## 20-3                 0                  0                   0
## 20-4                 0                  0                   0
## 20-5                 0                  0                   0
## 20-6                 0                  0                   0
## 20-7                 0                  0                   0
## 20-8                 0                  0                   0
## 21-1                 0                  0                   0
## 21-10                0                  0                   0
## 21-11                0                  0                   0
## 21-12                0                  0                   0
## 21-13                0                  0                   0
## 21-14                0                  0                   0
## 21-15                0                  0                   0
## 21-2                 0                  0                   0
## 21-3                 0                  0                   0
## 21-4                 0                  0                   0
## 21-5                 0                  0                   0
## 21-6                 0                  0                   0
## 21-7                 0                  0                   0
## 21-8                 0                  0                   0
## 21-9                 0                  0                   0
## 28-1                 0                  0                   0
## 28-10                0                  0                   0
## 28-11                0                  0                   0
## 28-12                0                  0                   0
## 28-13                0                  0                   0
## 28-14                0                  0                   0
## 28-15                0                  0                   0
## 28-2                 0                  0                   0
## 28-3                 0                  0                   0
## 28-4                 0                  0                   0
## 28-5                 0                  0                   0
## 28-6                 0                  0                   0
## 28-7                 0                  0                   0
## 28-8                 0                  0                   0
## 28-9                 0                  0                   0
## 29-1                 0                  0                   0
## 29-10                0                  0                   0
## 29-11                0                  0                   0
## 29-12                0                  0                   0
## 29-13                0                  0                   0
## 29-14                0                  0                   0
## 29-15                0                  0                   0
## 29-2                 0                  0                   0
## 29-3                 0                  0                   0
## 29-4                 0                  0                   0
## 29-5                 0                  0                   0
## 29-6                 0                  0                   0
## 29-7                 0                  0                   0
## 29-8                 0                  0                   0
## 29-9                 0                  0                   0
##       Rhodocytophaga sp. Oscillatoria sp..15 Aurantimonas sp..2
## 20-10                 61                  61                 60
## 20-11                  0                   0                  0
## 20-12                  0                   0                  0
## 20-13                  0                   0                  0
## 20-14                  0                   0                  0
## 20-15                  0                   0                  0
## 20-2                   0                   0                  0
## 20-3                   0                   0                  0
## 20-4                   0                   0                  0
## 20-5                   0                   0                  0
## 20-6                   0                   0                  0
## 20-7                   0                   0                  0
## 20-8                   0                   0                  0
## 21-1                   0                   0                  0
## 21-10                  0                   0                  0
## 21-11                  0                   0                  0
## 21-12                  0                   0                  0
## 21-13                  0                   0                  0
## 21-14                  0                   0                  0
## 21-15                  0                   0                  0
## 21-2                   0                   0                  0
## 21-3                   0                   0                  0
## 21-4                   0                   0                  0
## 21-5                   0                   0                  0
## 21-6                   0                   0                  0
## 21-7                   0                   0                  0
## 21-8                   0                   0                  0
## 21-9                   0                   0                  0
## 28-1                   0                   0                  0
## 28-10                  0                   0                  0
## 28-11                  0                   0                  0
## 28-12                  0                   0                  0
## 28-13                  0                   0                  0
## 28-14                  0                   0                  0
## 28-15                  0                   0                  0
## 28-2                   0                   0                  0
## 28-3                   0                   0                  0
## 28-4                   0                   0                  0
## 28-5                   0                   0                  0
## 28-6                   0                   0                  0
## 28-7                   0                   0                  0
## 28-8                   0                   0                  0
## 28-9                   0                   0                  0
## 29-1                   0                   0                  0
## 29-10                  0                   0                  0
## 29-11                  0                   0                  0
## 29-12                  0                   0                  0
## 29-13                  0                   0                  0
## 29-14                  0                   0                  0
## 29-15                  0                   0                  0
## 29-2                   0                   0                  0
## 29-3                   0                   0                  0
## 29-4                   0                   0                  0
## 29-5                   0                   0                  0
## 29-6                   0                   0                  0
## 29-7                   0                   0                  0
## 29-8                   0                   0                  0
## 29-9                   0                   0                  0
##       Granulicatella elegans Aureimonas sp..6 Sphingomonas sp..9
## 20-10                      0                0                  0
## 20-11                      0                0                  0
## 20-12                      0                0                  0
## 20-13                      0                0                  0
## 20-14                      0                0                  0
## 20-15                      0                0                  0
## 20-2                       0                0                  0
## 20-3                       0                0                  0
## 20-4                       0                0                  0
## 20-5                       0                0                  0
## 20-6                       0                0                  0
## 20-7                       0                0                  0
## 20-8                       0                0                  0
## 21-1                       0                0                  0
## 21-10                      0                0                  0
## 21-11                      0                0                  0
## 21-12                      0                0                  0
## 21-13                      0                0                  0
## 21-14                      0                0                  0
## 21-15                      0                0                  0
## 21-2                       0                0                  0
## 21-3                       0                0                  0
## 21-4                       0                0                  0
## 21-5                       0                0                  0
## 21-6                       0                0                  0
## 21-7                       0                0                  0
## 21-8                       0                0                  0
## 21-9                       0                0                  0
## 28-1                       0                0                  0
## 28-10                      0                0                  0
## 28-11                      0                0                  0
## 28-12                      0                0                  0
## 28-13                      0                0                  0
## 28-14                      0                0                  0
## 28-15                      0                0                  0
## 28-2                       0                0                  0
## 28-3                       0                0                  0
## 28-4                       0                0                  0
## 28-5                       0                0                  0
## 28-6                       0                0                  0
## 28-7                       0                0                  0
## 28-8                       0                0                  0
## 28-9                       0                0                  0
## 29-1                       0                0                  0
## 29-10                      0                0                  0
## 29-11                      0                0                  0
## 29-12                      0                0                  0
## 29-13                      0                0                  0
## 29-14                      0                0                 59
## 29-15                      0                0                  0
## 29-2                       0                0                  0
## 29-3                      60                0                  0
## 29-4                       0                0                  0
## 29-5                       0                0                  0
## 29-6                       0                0                  0
## 29-7                       0                0                  0
## 29-8                       0                0                  0
## 29-9                       0                0                  0
##       Kineococcus sp. Escherichia-Shigella sp..4 Oscillatoria sp..16
## 20-10               0                          0                   0
## 20-11               0                          0                   0
## 20-12               0                          0                   0
## 20-13               0                          0                   0
## 20-14               0                          0                   0
## 20-15               0                         58                   0
## 20-2                0                          0                   0
## 20-3                0                          0                   0
## 20-4                0                          0                   0
## 20-5                0                          0                   0
## 20-6                0                          0                   0
## 20-7                0                          0                   0
## 20-8                0                          0                   0
## 21-1                0                          0                   0
## 21-10               0                          0                   0
## 21-11               0                          0                   0
## 21-12               0                          0                   0
## 21-13               0                          0                   0
## 21-14               0                          0                   0
## 21-15               0                          0                   0
## 21-2                0                          0                   0
## 21-3                0                          0                   0
## 21-4                0                          0                   0
## 21-5                0                          0                   0
## 21-6                0                          0                   0
## 21-7                0                          0                   0
## 21-8                0                          0                   0
## 21-9                0                          0                   0
## 28-1                0                          0                   0
## 28-10               0                          0                   0
## 28-11               0                          0                   0
## 28-12               0                          0                   0
## 28-13               0                          0                   0
## 28-14               0                          0                   0
## 28-15               0                          0                   0
## 28-2                0                          0                   0
## 28-3                0                          0                   0
## 28-4                0                          0                   0
## 28-5                0                          0                   0
## 28-6                0                          0                   0
## 28-7                0                          0                   0
## 28-8                0                          0                   0
## 28-9                0                          0                   0
## 29-1                0                          0                   0
## 29-10               0                          0                   0
## 29-11               0                          0                   0
## 29-12               0                          0                   0
## 29-13               0                          0                   0
## 29-14               0                          0                   0
## 29-15               0                          0                   0
## 29-2                0                          0                   0
## 29-3                0                          0                   0
## 29-4                0                          0                   0
## 29-5                0                          0                   0
## 29-6                0                          0                   0
## 29-7                0                          0                   0
## 29-8                0                          0                   0
## 29-9                0                          0                   0
##       Oscillatoria sp..17 Methylobacterium-Methylorubrum sp..38
## 20-10                   0                                     0
## 20-11                   0                                     0
## 20-12                   0                                     0
## 20-13                   0                                     0
## 20-14                   0                                     0
## 20-15                   0                                     0
## 20-2                    0                                     0
## 20-3                    0                                     0
## 20-4                    0                                     0
## 20-5                    0                                     0
## 20-6                    0                                     0
## 20-7                    0                                     0
## 20-8                    0                                     0
## 21-1                    0                                     0
## 21-10                   0                                     0
## 21-11                   0                                     0
## 21-12                   0                                     0
## 21-13                   0                                     0
## 21-14                   0                                     0
## 21-15                   0                                     0
## 21-2                    0                                     0
## 21-3                    0                                     0
## 21-4                    0                                     0
## 21-5                    0                                     0
## 21-6                    0                                     0
## 21-7                    0                                     0
## 21-8                    0                                     0
## 21-9                    0                                     0
## 28-1                    0                                     0
## 28-10                   0                                     0
## 28-11                   0                                     0
## 28-12                   0                                     0
## 28-13                  57                                     0
## 28-14                   0                                     0
## 28-15                   0                                     0
## 28-2                    0                                     0
## 28-3                    0                                     0
## 28-4                    0                                     0
## 28-5                    0                                     0
## 28-6                    0                                     0
## 28-7                    0                                     0
## 28-8                    0                                     0
## 28-9                    0                                    54
## 29-1                    0                                     0
## 29-10                   0                                     0
## 29-11                   0                                     0
## 29-12                   0                                     0
## 29-13                   0                                     0
## 29-14                   0                                     0
## 29-15                   0                                     0
## 29-2                    0                                     0
## 29-3                    0                                     0
## 29-4                    0                                     0
## 29-5                    0                                     0
## 29-6                    0                                     0
## 29-7                    0                                     0
## 29-8                    0                                     0
## 29-9                    0                                     0
##       Oscillatoria sp..18 Methylobacterium-Methylorubrum sp..39
## 20-10                   0                                     0
## 20-11                   0                                     0
## 20-12                   0                                     0
## 20-13                   0                                     0
## 20-14                   0                                     0
## 20-15                   0                                     0
## 20-2                    0                                     0
## 20-3                    0                                     0
## 20-4                    0                                     0
## 20-5                    0                                     0
## 20-6                    0                                     0
## 20-7                    0                                     0
## 20-8                    0                                     0
## 21-1                    0                                    53
## 21-10                   0                                     0
## 21-11                   0                                     0
## 21-12                   0                                     0
## 21-13                   0                                     0
## 21-14                   0                                     0
## 21-15                   0                                     0
## 21-2                    0                                     0
## 21-3                    0                                     0
## 21-4                    0                                     0
## 21-5                    0                                     0
## 21-6                    0                                     0
## 21-7                    0                                     0
## 21-8                    0                                     0
## 21-9                    0                                     0
## 28-1                    0                                     0
## 28-10                   0                                     0
## 28-11                   0                                     0
## 28-12                   0                                     0
## 28-13                   0                                     0
## 28-14                   0                                     0
## 28-15                   0                                     0
## 28-2                    0                                     0
## 28-3                    0                                     0
## 28-4                    0                                     0
## 28-5                    0                                     0
## 28-6                    0                                     0
## 28-7                    0                                     0
## 28-8                    0                                     0
## 28-9                    0                                     0
## 29-1                    0                                     0
## 29-10                   0                                     0
## 29-11                   0                                     0
## 29-12                   0                                     0
## 29-13                   0                                     0
## 29-14                   0                                     0
## 29-15                   0                                     0
## 29-2                    0                                     0
## 29-3                    0                                     0
## 29-4                    0                                     0
## 29-5                    0                                     0
## 29-6                    0                                     0
## 29-7                    0                                     0
## 29-8                    0                                     0
## 29-9                    0                                     0
##       Aureimonas sp..7 Staphylococcus sp. Aureimonas sp..8 Enterobacteriaceae
## 20-10                0                  0                0                  0
## 20-11                0                  0                0                  0
## 20-12                0                  0                0                  0
## 20-13                0                  0                0                  0
## 20-14                0                  0                0                  0
## 20-15                0                  0                0                  0
## 20-2                 0                  0                0                  0
## 20-3                 0                  0                0                  0
## 20-4                 0                  0                0                  0
## 20-5                 0                  0                0                  0
## 20-6                 0                  0                0                  0
## 20-7                 0                  0                0                  0
## 20-8                 0                  0                0                  0
## 21-1                 0                  0                0                  0
## 21-10                0                  0                0                  0
## 21-11                0                  0                0                  0
## 21-12                0                  0                0                  0
## 21-13                0                  0                0                  0
## 21-14                0                  0                0                  0
## 21-15                0                  0                0                  0
## 21-2                 0                  0                0                  0
## 21-3                 0                  0                0                  0
## 21-4                 0                  0                0                  0
## 21-5                 0                  0                0                  0
## 21-6                 0                  0                0                  0
## 21-7                 0                  0                0                  0
## 21-8                 0                  0                0                  0
## 21-9                 0                  0                0                  0
## 28-1                 0                  0                0                  0
## 28-10                0                  0                0                  0
## 28-11                0                  0               51                  0
## 28-12                0                  0                0                  0
## 28-13                0                  0                0                  0
## 28-14                0                  0                0                  0
## 28-15               52                  0                0                  0
## 28-2                 0                  0                0                  0
## 28-3                 0                  0                0                  0
## 28-4                 0                 25                0                  0
## 28-5                 0                  0                0                  0
## 28-6                 0                  0                0                  0
## 28-7                 0                  0                0                  0
## 28-8                 0                  0                0                  0
## 28-9                 0                  0                0                  0
## 29-1                 0                  0                0                  0
## 29-10                0                  0                0                  0
## 29-11                0                  0                0                  0
## 29-12                0                  0                0                  0
## 29-13                0                  0                0                  0
## 29-14                0                  0                0                  0
## 29-15                0                  0                0                  0
## 29-2                 0                  0                0                  0
## 29-3                 0                  0                0                  0
## 29-4                 0                  0                0                  0
## 29-5                 0                  0                0                  0
## 29-6                 0                  0                0                  0
## 29-7                 0                  0                0                  0
## 29-8                 0                  0                0                  0
## 29-9                 0                  0                0                  0
##       Oscillatoria sp..19 Hymenobacter sp..6 Sphingomonas sp..10
## 20-10                   0                  0                   0
## 20-11                   0                  0                   0
## 20-12                   0                  0                   0
## 20-13                   0                  0                   0
## 20-14                   0                  0                   0
## 20-15                   0                  0                   0
## 20-2                    0                  0                   0
## 20-3                    0                  0                   0
## 20-4                    0                  0                   0
## 20-5                    0                  0                   0
## 20-6                    0                  0                   0
## 20-7                    0                  0                   0
## 20-8                    0                  0                   0
## 21-1                    0                  0                   0
## 21-10                   0                  0                   0
## 21-11                   0                  0                   0
## 21-12                   0                  0                   0
## 21-13                  48                  0                   0
## 21-14                   0                  0                   0
## 21-15                   0                  0                   0
## 21-2                    0                  0                   0
## 21-3                    0                  0                   0
## 21-4                    0                  0                  46
## 21-5                    0                  0                   0
## 21-6                    0                  0                   0
## 21-7                    0                  0                   0
## 21-8                    0                  0                   0
## 21-9                    0                  0                   0
## 28-1                    0                  0                   0
## 28-10                   0                  0                   0
## 28-11                   0                  0                   0
## 28-12                   0                  0                   0
## 28-13                   0                  0                   0
## 28-14                   0                  0                   0
## 28-15                   0                  0                   0
## 28-2                    0                  0                   0
## 28-3                    0                  0                   0
## 28-4                    0                  0                   0
## 28-5                    0                  0                   0
## 28-6                    0                  0                   0
## 28-7                    0                  0                   0
## 28-8                    0                  0                   0
## 28-9                    0                  0                   0
## 29-1                    0                  0                   0
## 29-10                   0                  0                   0
## 29-11                   0                  0                   0
## 29-12                   0                  0                   0
## 29-13                   0                  0                   0
## 29-14                   0                 48                   0
## 29-15                   0                  0                   0
## 29-2                    0                  0                   0
## 29-3                    0                  0                   0
## 29-4                    0                  0                   0
## 29-5                    0                  0                   0
## 29-6                    0                  0                   0
## 29-7                    0                  0                   0
## 29-8                    0                  0                   0
## 29-9                    0                  0                   0
##       Oscillatoria sp..20 Gemmatimonadaceae Spirosoma sp. Oscillatoria sp..21
## 20-10                   0                 0             0                   0
## 20-11                   0                 0             0                   0
## 20-12                   0                 0             0                   0
## 20-13                   0                 0             0                   0
## 20-14                   0                 0             0                   0
## 20-15                   0                 0             0                   0
## 20-2                    0                 0             0                   0
## 20-3                    0                 0             0                   0
## 20-4                    0                 0             0                   0
## 20-5                    0                 0             0                   0
## 20-6                    0                 0             0                   0
## 20-7                    0                 0             0                   0
## 20-8                    0                 0             0                   0
## 21-1                    0                 0             0                   0
## 21-10                   0                 0             0                   0
## 21-11                   0                 0             0                   0
## 21-12                   0                 0             0                   0
## 21-13                   0                 0             0                   0
## 21-14                   0                 0             0                   0
## 21-15                   0                 0             0                   0
## 21-2                    0                 0             0                   0
## 21-3                    0                 0             0                   0
## 21-4                    0                 0             0                   0
## 21-5                    0                 0             0                   0
## 21-6                    0                 0             0                   0
## 21-7                    0                 0             0                   0
## 21-8                    0                 0             0                   0
## 21-9                    0                 0             0                   0
## 28-1                    0                 0             0                   0
## 28-10                   0                 0             0                   0
## 28-11                   0                 0             0                   0
## 28-12                   0                 0             0                   0
## 28-13                   0                 0             0                   0
## 28-14                   0                 0             0                   0
## 28-15                   0                 0             0                   0
## 28-2                    0                 0             0                   0
## 28-3                    0                 0             0                   0
## 28-4                    0                 0             0                   0
## 28-5                    0                 0             0                   0
## 28-6                    0                 0             0                   0
## 28-7                    0                 0             0                   0
## 28-8                    0                 0             0                   0
## 28-9                   46                 0             0                   0
## 29-1                    0                 0             0                   0
## 29-10                   0                 0             0                   0
## 29-11                   0                 0             0                   0
## 29-12                   0                 0             0                   0
## 29-13                   0                 0             0                   0
## 29-14                   0                 0             0                   0
## 29-15                   0                 0             0                   0
## 29-2                    0                 0             0                   0
## 29-3                    0                 0             0                   0
## 29-4                    0                 0             0                   0
## 29-5                    0                 0             0                   0
## 29-6                    0                 0             0                   0
## 29-7                    0                 0             0                   0
## 29-8                    0                 0            25                   0
## 29-9                    0                46             0                   0
##       Methylobacterium-Methylorubrum sp..40 Oscillatoria sp..22
## 20-10                                     0                   0
## 20-11                                     0                   0
## 20-12                                     0                   0
## 20-13                                     0                   0
## 20-14                                     0                   0
## 20-15                                     0                   0
## 20-2                                      0                   0
## 20-3                                      0                   0
## 20-4                                      0                   0
## 20-5                                      0                   0
## 20-6                                      0                   0
## 20-7                                      0                   0
## 20-8                                      0                   0
## 21-1                                      0                   0
## 21-10                                     0                   0
## 21-11                                     0                   0
## 21-12                                     0                   0
## 21-13                                     0                   0
## 21-14                                     0                   0
## 21-15                                     0                   0
## 21-2                                      0                   0
## 21-3                                      0                   0
## 21-4                                      0                   0
## 21-5                                      0                   0
## 21-6                                      0                   0
## 21-7                                      0                   0
## 21-8                                      0                   0
## 21-9                                      0                   0
## 28-1                                      0                   0
## 28-10                                     0                   0
## 28-11                                     0                   0
## 28-12                                     0                   0
## 28-13                                     0                   0
## 28-14                                     0                  44
## 28-15                                     0                   0
## 28-2                                      0                   0
## 28-3                                      0                   0
## 28-4                                      0                   0
## 28-5                                      0                   0
## 28-6                                      0                   0
## 28-7                                      0                   0
## 28-8                                      0                   0
## 28-9                                      0                   0
## 29-1                                      0                   0
## 29-10                                     0                   0
## 29-11                                     0                   0
## 29-12                                     0                   0
## 29-13                                     0                   0
## 29-14                                     0                   0
## 29-15                                     0                   0
## 29-2                                      0                   0
## 29-3                                      0                   0
## 29-4                                      0                   0
## 29-5                                      0                   0
## 29-6                                      0                   0
## 29-7                                      0                   0
## 29-8                                      0                   0
## 29-9                                      0                   0
##       Oscillatoria sp..23 Oscillatoria sp..24
## 20-10                   0                   0
## 20-11                   0                   0
## 20-12                   0                   0
## 20-13                   0                   0
## 20-14                   0                   0
## 20-15                   0                   0
## 20-2                    0                   0
## 20-3                    0                   0
## 20-4                    0                   0
## 20-5                    0                   0
## 20-6                    0                   0
## 20-7                    0                   0
## 20-8                    0                   0
## 21-1                    0                   0
## 21-10                   0                   0
## 21-11                   0                   0
## 21-12                   0                   0
## 21-13                   0                   0
## 21-14                   0                   0
## 21-15                   0                   0
## 21-2                    0                   0
## 21-3                    0                   0
## 21-4                    0                   0
## 21-5                    0                   0
## 21-6                    0                   0
## 21-7                    0                   0
## 21-8                    0                   0
## 21-9                    0                   0
## 28-1                    0                   0
## 28-10                   0                   0
## 28-11                   0                   0
## 28-12                   0                   0
## 28-13                   0                   0
## 28-14                   0                   0
## 28-15                   0                   0
## 28-2                    0                   0
## 28-3                    0                   0
## 28-4                    0                   0
## 28-5                    0                   0
## 28-6                    0                   0
## 28-7                    0                   0
## 28-8                    0                   0
## 28-9                    0                   0
## 29-1                    0                   0
## 29-10                   0                   0
## 29-11                   0                   0
## 29-12                   0                   0
## 29-13                   0                   0
## 29-14                   0                   0
## 29-15                   0                   0
## 29-2                    0                   0
## 29-3                    0                   0
## 29-4                    0                   0
## 29-5                    0                   0
## 29-6                    0                   0
## 29-7                    0                   0
## 29-8                    0                   0
## 29-9                    0                   0
##       Methylobacterium-Methylorubrum sp..41
## 20-10                                     0
## 20-11                                     0
## 20-12                                     0
## 20-13                                     0
## 20-14                                     0
## 20-15                                     0
## 20-2                                      0
## 20-3                                      0
## 20-4                                      0
## 20-5                                      0
## 20-6                                      0
## 20-7                                      0
## 20-8                                      0
## 21-1                                      0
## 21-10                                     0
## 21-11                                     0
## 21-12                                     0
## 21-13                                     0
## 21-14                                     0
## 21-15                                     0
## 21-2                                      0
## 21-3                                      0
## 21-4                                      0
## 21-5                                      0
## 21-6                                      0
## 21-7                                      0
## 21-8                                      0
## 21-9                                      0
## 28-1                                      0
## 28-10                                     0
## 28-11                                     0
## 28-12                                     0
## 28-13                                     0
## 28-14                                     0
## 28-15                                     0
## 28-2                                      0
## 28-3                                      0
## 28-4                                      0
## 28-5                                      0
## 28-6                                      0
## 28-7                                      0
## 28-8                                      0
## 28-9                                      0
## 29-1                                      0
## 29-10                                     0
## 29-11                                     0
## 29-12                                     0
## 29-13                                     0
## 29-14                                     0
## 29-15                                     0
## 29-2                                      0
## 29-3                                      0
## 29-4                                      0
## 29-5                                      0
## 29-6                                      0
## 29-7                                      0
## 29-8                                      0
## 29-9                                      0
##       Methylobacterium-Methylorubrum sp..42 Aureimonas sp..9
## 20-10                                     0                0
## 20-11                                     0                0
## 20-12                                     0                0
## 20-13                                     0                0
## 20-14                                     0                0
## 20-15                                     0                0
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## 21-10                                     0                0
## 21-11                                     0                0
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## 21-13                                     0                0
## 21-14                                     0                0
## 21-15                                     0                0
## 21-2                                      0                0
## 21-3                                      0                0
## 21-4                                      0                0
## 21-5                                      0                0
## 21-6                                      0                0
## 21-7                                      0                0
## 21-8                                      0                0
## 21-9                                      0                0
## 28-1                                      0                0
## 28-10                                     0                0
## 28-11                                     0                0
## 28-12                                     0                0
## 28-13                                     0                0
## 28-14                                     0               38
## 28-15                                     0                0
## 28-2                                      0                0
## 28-3                                      0                0
## 28-4                                      0                0
## 28-5                                      0                0
## 28-6                                      0                0
## 28-7                                      0                0
## 28-8                                      0                0
## 28-9                                      0                0
## 29-1                                      0                0
## 29-10                                     0                0
## 29-11                                    40                0
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## 29-5                                      0                0
## 29-6                                      0                0
## 29-7                                      0                0
## 29-8                                      0                0
## 29-9                                      0                0
##       Microbacterium sp..1 Oscillatoria sp..25 Ramlibacter sp.
## 20-10                    0                   0               0
## 20-11                    0                   0               0
## 20-12                    0                   0               0
## 20-13                    0                   0               0
## 20-14                    0                   0               0
## 20-15                    0                   0               0
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## 21-12                    0                   0               0
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## 21-2                     0                   0               0
## 21-3                     0                   0               0
## 21-4                    37                   0               0
## 21-5                     0                   0               0
## 21-6                     0                   0               0
## 21-7                     0                   0               0
## 21-8                     0                   0               0
## 21-9                     0                   0               0
## 28-1                     0                   0               0
## 28-10                    0                   0               0
## 28-11                    0                   0               0
## 28-12                    0                   0               0
## 28-13                    0                   0               0
## 28-14                    0                   0               0
## 28-15                    0                   0               0
## 28-2                     0                   0               0
## 28-3                     0                   0               0
## 28-4                     0                   0               0
## 28-5                     0                   0               0
## 28-6                     0                   0               0
## 28-7                     0                   0               0
## 28-8                     0                   0               0
## 28-9                     0                   0               0
## 29-1                     0                   0               0
## 29-10                    0                   0               0
## 29-11                    0                   0               0
## 29-12                    0                   0               0
## 29-13                    0                   0               0
## 29-14                    0                   0               0
## 29-15                    0                   0               0
## 29-2                     0                   0               0
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## 29-5                     0                   0               0
## 29-6                     0                   0               0
## 29-7                     0                   0               0
## 29-8                     0                   0               0
## 29-9                     0                   0               0
##       Aureimonas sp..10 Methylobacterium-Methylorubrum sp..43 Aureimonas sp..11
## 20-10                 0                                     0                 0
## 20-11                 0                                     0                 0
## 20-12                 0                                     0                 0
## 20-13                 0                                     0                 0
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## 20-3                  0                                     0                 0
## 20-4                  0                                     0                 0
## 20-5                  0                                     0                 0
## 20-6                  0                                     0                 0
## 20-7                  0                                     0                 0
## 20-8                  0                                     0                 0
## 21-1                  0                                     0                 0
## 21-10                 0                                     0                 0
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## 21-3                  0                                     0                 0
## 21-4                  0                                     0                 0
## 21-5                  0                                     0                 0
## 21-6                  0                                     0                 0
## 21-7                  0                                     0                 0
## 21-8                  0                                     0                 0
## 21-9                  0                                     0                 0
## 28-1                  0                                     0                 0
## 28-10                 0                                     0                 0
## 28-11                 0                                     0                 0
## 28-12                 0                                     0                 0
## 28-13                 0                                     0                 0
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## 28-15                 0                                     0                 0
## 28-2                  0                                     0                 0
## 28-3                  0                                     0                 0
## 28-4                  0                                     0                 0
## 28-5                  0                                     0                 0
## 28-6                  0                                     0                 0
## 28-7                 37                                     0                 0
## 28-8                  0                                     0                 0
## 28-9                  0                                     0                 0
## 29-1                  0                                     0                 0
## 29-10                 0                                     0                 0
## 29-11                 0                                     0                 0
## 29-12                 0                                     0                 0
## 29-13                 0                                     0                 0
## 29-14                 0                                     0                 0
## 29-15                 0                                     0                 0
## 29-2                  0                                     0                 0
## 29-3                  0                                     0                 0
## 29-4                  0                                     0                 0
## 29-5                  0                                     0                 0
## 29-6                  0                                     0                 0
## 29-7                  0                                     0                 0
## 29-8                  0                                     0                 0
## 29-9                  0                                     0                 0
##       Oscillatoria sp..26 Hymenobacter sp..7 Oscillatoria sp..27
## 20-10                   0                  0                   0
## 20-11                   0                  0                   0
## 20-12                   0                  0                   0
## 20-13                   0                  0                   0
## 20-14                   0                  0                   0
## 20-15                   0                  0                   0
## 20-2                    0                  0                   0
## 20-3                    0                  0                   0
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## 21-10                   0                  0                   0
## 21-11                   0                  0                   0
## 21-12                   0                  0                   0
## 21-13                   0                  0                   0
## 21-14                   0                  0                   0
## 21-15                   0                  0                   0
## 21-2                    0                  0                   0
## 21-3                    0                  0                   0
## 21-4                    0                  0                   0
## 21-5                    0                  0                   0
## 21-6                    0                  0                   0
## 21-7                    0                  0                   0
## 21-8                    0                 33                   0
## 21-9                    0                  0                   0
## 28-1                    0                  0                   0
## 28-10                   0                  0                   0
## 28-11                   0                  0                   0
## 28-12                   0                  0                   0
## 28-13                   0                  0                   0
## 28-14                   0                  0                   0
## 28-15                  34                  0                   0
## 28-2                    0                  0                   0
## 28-3                    0                  0                   0
## 28-4                    0                  0                   0
## 28-5                    0                  0                   0
## 28-6                    0                  0                   0
## 28-7                    0                  0                   0
## 28-8                    0                  0                   0
## 28-9                    0                  0                   0
## 29-1                    0                  0                   0
## 29-10                   0                  0                   0
## 29-11                   0                  0                   0
## 29-12                   0                  0                   0
## 29-13                   0                  0                   0
## 29-14                   0                  0                  33
## 29-15                   0                  0                   0
## 29-2                    0                  0                   0
## 29-3                    0                  0                   0
## 29-4                    0                  0                   0
## 29-5                    0                  0                   0
## 29-6                    0                  0                   0
## 29-7                    0                  0                   0
## 29-8                    0                  0                   0
## 29-9                    0                  0                   0
##       Oscillatoria sp..28 Oscillatoria sp..29 Quadrisphaera sp..3
## 20-10                   0                   0                   0
## 20-11                   0                   0                   0
## 20-12                   0                   0                   0
## 20-13                   0                   0                   0
## 20-14                   0                   0                   0
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## 20-3                    0                   0                   0
## 20-4                    0                   0                   0
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## 21-10                   0                   0                   0
## 21-11                   0                   0                   0
## 21-12                   0                   0                   0
## 21-13                   0                   0                   0
## 21-14                   0                   0                   0
## 21-15                   0                   0                   0
## 21-2                    0                   0                   0
## 21-3                    0                   0                   0
## 21-4                    0                   0                   0
## 21-5                   32                   0                   0
## 21-6                    0                   0                   0
## 21-7                    0                   0                   0
## 21-8                    0                   0                   0
## 21-9                    0                   0                   0
## 28-1                    0                   0                   0
## 28-10                   0                   0                   0
## 28-11                   0                   0                   0
## 28-12                   0                   0                   0
## 28-13                   0                   0                   0
## 28-14                   0                   0                   0
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## 28-3                    0                   0                   0
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## 28-5                    0                   0                   0
## 28-6                    0                   0                   0
## 28-7                    0                   0                   0
## 28-8                    0                   0                   0
## 28-9                    0                   0                   0
## 29-1                    0                   0                   0
## 29-10                   0                   0                   0
## 29-11                   0                   0                   0
## 29-12                   0                   0                   0
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## 29-14                   0                   0                   0
## 29-15                   0                   0                   0
## 29-2                    0                   0                   0
## 29-3                    0                   0                   0
## 29-4                    0                   0                   0
## 29-5                    0                   0                   0
## 29-6                    0                   0                   0
## 29-7                    0                   0                   0
## 29-8                    0                   0                   0
## 29-9                    0                   0                   0
##       Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium sp.
## 20-10                                                      0
## 20-11                                                      0
## 20-12                                                      0
## 20-13                                                      0
## 20-14                                                      0
## 20-15                                                      0
## 20-2                                                       0
## 20-3                                                       0
## 20-4                                                       0
## 20-5                                                       0
## 20-6                                                       0
## 20-7                                                       0
## 20-8                                                       0
## 21-1                                                       0
## 21-10                                                      0
## 21-11                                                      0
## 21-12                                                      0
## 21-13                                                      0
## 21-14                                                      0
## 21-15                                                      0
## 21-2                                                       0
## 21-3                                                       0
## 21-4                                                       0
## 21-5                                                       0
## 21-6                                                       0
## 21-7                                                       0
## 21-8                                                       0
## 21-9                                                       0
## 28-1                                                       0
## 28-10                                                      0
## 28-11                                                      0
## 28-12                                                      0
## 28-13                                                      0
## 28-14                                                      0
## 28-15                                                      0
## 28-2                                                       0
## 28-3                                                       0
## 28-4                                                       0
## 28-5                                                       0
## 28-6                                                       0
## 28-7                                                       0
## 28-8                                                       0
## 28-9                                                       0
## 29-1                                                       0
## 29-10                                                      0
## 29-11                                                      0
## 29-12                                                      0
## 29-13                                                      0
## 29-14                                                      0
## 29-15                                                      0
## 29-2                                                       0
## 29-3                                                       0
## 29-4                                                       0
## 29-5                                                       0
## 29-6                                                       0
## 29-7                                                       0
## 29-8                                                       0
## 29-9                                                       0
##       Oscillatoria sp..30 Methylobacterium-Methylorubrum sp..44 Klebsiella sp.
## 20-10                   0                                     0              0
## 20-11                   0                                     0              0
## 20-12                   0                                     0              0
## 20-13                   0                                     0              0
## 20-14                   0                                     0              0
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## 20-8                    0                                     0              0
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## 21-10                   0                                     0              0
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## 21-7                    0                                     0              0
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## 21-9                    0                                     0              0
## 28-1                    0                                     0              0
## 28-10                   0                                     0              0
## 28-11                   0                                    31              0
## 28-12                   0                                     0              0
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## 28-6                    0                                     0              0
## 28-7                    0                                     0              0
## 28-8                    0                                     0              0
## 28-9                    0                                     0              0
## 29-1                    0                                     0              0
## 29-10                   0                                     0              0
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## 29-12                   0                                     0              0
## 29-13                   0                                     0              0
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## 29-3                    0                                     0              0
## 29-4                    0                                     0              0
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## 29-6                    0                                     0              0
## 29-7                    0                                     0              0
## 29-8                    0                                     0              0
## 29-9                    0                                     0              0
##       Methylobacterium-Methylorubrum sp..45 Oscillatoria sp..31
## 20-10                                     0                   0
## 20-11                                     0                   0
## 20-12                                     0                   0
## 20-13                                     0                   0
## 20-14                                     0                   0
## 20-15                                     0                   0
## 20-2                                      0                   0
## 20-3                                      0                   0
## 20-4                                      0                  29
## 20-5                                      0                   0
## 20-6                                      0                   0
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## 21-1                                      0                   0
## 21-10                                     0                   0
## 21-11                                     0                   0
## 21-12                                     0                   0
## 21-13                                     0                   0
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## 21-15                                     0                   0
## 21-2                                      0                   0
## 21-3                                      0                   0
## 21-4                                      0                   0
## 21-5                                      0                   0
## 21-6                                      0                   0
## 21-7                                      0                   0
## 21-8                                      0                   0
## 21-9                                      0                   0
## 28-1                                      0                   0
## 28-10                                     0                   0
## 28-11                                     0                   0
## 28-12                                     0                   0
## 28-13                                     0                   0
## 28-14                                     0                   0
## 28-15                                     0                   0
## 28-2                                      0                   0
## 28-3                                      0                   0
## 28-4                                      0                   0
## 28-5                                      0                   0
## 28-6                                      0                   0
## 28-7                                      0                   0
## 28-8                                      0                   0
## 28-9                                      0                   0
## 29-1                                      0                   0
## 29-10                                     0                   0
## 29-11                                     0                   0
## 29-12                                     0                   0
## 29-13                                     0                   0
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## 29-5                                      0                   0
## 29-6                                      0                   0
## 29-7                                     30                   0
## 29-8                                      0                   0
## 29-9                                      0                   0
##       Nocardioides alpinus Methylobacterium-Methylorubrum sp..46
## 20-10                    0                                     0
## 20-11                    0                                     0
## 20-12                    0                                     0
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## 20-5                    29                                     0
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## 21-10                    0                                     0
## 21-11                    0                                     0
## 21-12                    0                                     0
## 21-13                    0                                     0
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## 21-15                    0                                     0
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## 28-10                    0                                     0
## 28-11                    0                                     0
## 28-12                    0                                     0
## 28-13                    0                                     0
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## 28-15                    0                                     0
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## 28-9                     0                                     0
## 29-1                     0                                     0
## 29-10                    0                                     0
## 29-11                    0                                     0
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## 29-13                    0                                     0
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## 29-15                    0                                     0
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## 29-5                     0                                     0
## 29-6                     0                                     0
## 29-7                     0                                     0
## 29-8                     0                                     0
## 29-9                     0                                     0
##       Oscillatoria sp..32 Escherichia-Shigella sp..5 Oscillatoria sp..33
## 20-10                   0                          0                   0
## 20-11                   0                          0                   0
## 20-12                   0                          0                   0
## 20-13                   0                          0                   0
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## 28-5                    0                          0                   0
## 28-6                    0                          0                   0
## 28-7                    0                          0                  27
## 28-8                    0                          0                   0
## 28-9                    0                          0                   0
## 29-1                    0                          0                   0
## 29-10                   0                          0                   0
## 29-11                   0                          0                   0
## 29-12                   0                          0                   0
## 29-13                   0                          0                   0
## 29-14                   0                          0                   0
## 29-15                   0                          0                   0
## 29-2                    0                          0                   0
## 29-3                    0                          0                   0
## 29-4                    0                          0                   0
## 29-5                    0                          0                   0
## 29-6                    0                          0                   0
## 29-7                    0                          0                   0
## 29-8                    0                          0                   0
## 29-9                    0                          0                   0
##       Sphingomonas sp..11 Bdellovibrio sp..1
## 20-10                   0                  0
## 20-11                   0                  0
## 20-12                   0                  0
## 20-13                   0                  0
## 20-14                   0                  0
## 20-15                   0                  0
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## 20-7                    0                  0
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## 21-1                    0                  0
## 21-10                   0                  0
## 21-11                   0                  0
## 21-12                   0                  0
## 21-13                   0                  0
## 21-14                   0                  0
## 21-15                   0                  0
## 21-2                    0                  0
## 21-3                    0                  0
## 21-4                    0                  0
## 21-5                    0                  0
## 21-6                    0                  0
## 21-7                    0                  0
## 21-8                    0                  0
## 21-9                    0                  0
## 28-1                    0                  0
## 28-10                   0                  0
## 28-11                   0                  0
## 28-12                   0                  0
## 28-13                   0                  0
## 28-14                   0                  0
## 28-15                   0                  0
## 28-2                    0                  0
## 28-3                    0                  0
## 28-4                    0                  0
## 28-5                    0                  0
## 28-6                    0                  0
## 28-7                    0                  0
## 28-8                    0                  0
## 28-9                    0                  0
## 29-1                    0                  0
## 29-10                   0                  0
## 29-11                   0                  0
## 29-12                   0                  0
## 29-13                  27                  0
## 29-14                   0                  0
## 29-15                   0                  0
## 29-2                    0                  0
## 29-3                    0                  0
## 29-4                    0                 24
## 29-5                    0                  0
## 29-6                    0                  0
## 29-7                    0                  0
## 29-8                    0                  0
## 29-9                    0                  0
##       Methylobacterium-Methylorubrum sp..47 Escherichia-Shigella sp..6
## 20-10                                     0                          0
## 20-11                                     0                          0
## 20-12                                     0                          0
## 20-13                                     0                          0
## 20-14                                     0                          0
## 20-15                                     0                          0
## 20-2                                      0                          0
## 20-3                                      0                          0
## 20-4                                      0                          0
## 20-5                                      0                          0
## 20-6                                      0                          0
## 20-7                                      0                          0
## 20-8                                      0                          0
## 21-1                                      0                          0
## 21-10                                     0                          0
## 21-11                                     0                          0
## 21-12                                     0                          0
## 21-13                                     0                          0
## 21-14                                     0                          0
## 21-15                                     0                          0
## 21-2                                      0                          0
## 21-3                                      0                          0
## 21-4                                      0                          0
## 21-5                                      0                          0
## 21-6                                      0                          0
## 21-7                                      0                          0
## 21-8                                      0                          0
## 21-9                                      0                          0
## 28-1                                      0                          0
## 28-10                                     0                          0
## 28-11                                     0                          0
## 28-12                                     0                          0
## 28-13                                     0                          0
## 28-14                                     0                          0
## 28-15                                     0                          0
## 28-2                                      0                          0
## 28-3                                      0                          0
## 28-4                                      0                          0
## 28-5                                      0                          0
## 28-6                                      0                          0
## 28-7                                      0                          0
## 28-8                                      0                          0
## 28-9                                      0                          0
## 29-1                                      0                          0
## 29-10                                     0                          0
## 29-11                                     0                          0
## 29-12                                     0                          0
## 29-13                                     0                          0
## 29-14                                     0                          0
## 29-15                                     0                          0
## 29-2                                      0                          0
## 29-3                                      0                          0
## 29-4                                     24                          0
## 29-5                                      0                          0
## 29-6                                      0                          0
## 29-7                                      0                          0
## 29-8                                      0                          0
## 29-9                                      0                          0
##       Oscillatoria sp..34 Oscillatoria sp..35 Hymenobacter sp..8
## 20-10                   0                   0                  0
## 20-11                   0                   0                  0
## 20-12                   0                   0                  0
## 20-13                   0                   0                  0
## 20-14                   0                   0                  0
## 20-15                   0                   0                  0
## 20-2                    0                   0                  0
## 20-3                    0                   0                  0
## 20-4                    0                   0                  0
## 20-5                    0                   0                  0
## 20-6                    0                   0                  0
## 20-7                    0                   0                  0
## 20-8                    0                   0                  0
## 21-1                    0                   0                  0
## 21-10                  23                   0                  0
## 21-11                   0                   0                 22
## 21-12                   0                   0                  0
## 21-13                   0                   0                  0
## 21-14                   0                   0                  0
## 21-15                   0                   0                  0
## 21-2                    0                   0                  0
## 21-3                    0                   0                  0
## 21-4                    0                   0                  0
## 21-5                    0                   0                  0
## 21-6                    0                   0                  0
## 21-7                    0                   0                  0
## 21-8                    0                   0                  0
## 21-9                    0                   0                  0
## 28-1                    0                   0                  0
## 28-10                   0                   0                  0
## 28-11                   0                   0                  0
## 28-12                   0                   0                  0
## 28-13                   0                   0                  0
## 28-14                   0                   0                  0
## 28-15                   0                   0                  0
## 28-2                    0                   0                  0
## 28-3                    0                   0                  0
## 28-4                    0                   0                  0
## 28-5                    0                   0                  0
## 28-6                    0                   0                  0
## 28-7                    0                   0                  0
## 28-8                    0                   0                  0
## 28-9                    0                   0                  0
## 29-1                    0                   0                  0
## 29-10                   0                   0                  0
## 29-11                   0                   0                  0
## 29-12                   0                   0                  0
## 29-13                   0                   0                  0
## 29-14                   0                   0                  0
## 29-15                   0                   0                  0
## 29-2                    0                   0                  0
## 29-3                    0                   0                  0
## 29-4                    0                   0                  0
## 29-5                    0                   0                  0
## 29-6                    0                  23                  0
## 29-7                    0                   0                  0
## 29-8                    0                   0                  0
## 29-9                    0                   0                  0
##       Methylobacterium-Methylorubrum sp..48 Mesorhizobium sp.
## 20-10                                     0                 0
## 20-11                                     0                 0
## 20-12                                     0                 0
## 20-13                                     0                 0
## 20-14                                     0                 0
## 20-15                                     0                 0
## 20-2                                      0                 0
## 20-3                                      0                 0
## 20-4                                      0                 0
## 20-5                                      0                 0
## 20-6                                      0                 0
## 20-7                                      0                 0
## 20-8                                      0                 0
## 21-1                                      0                 0
## 21-10                                     0                 0
## 21-11                                     0                 0
## 21-12                                     0                 0
## 21-13                                     0                 0
## 21-14                                     0                 0
## 21-15                                     0                 0
## 21-2                                      0                 0
## 21-3                                      0                 0
## 21-4                                      0                 0
## 21-5                                      0                 0
## 21-6                                      0                 0
## 21-7                                      0                 0
## 21-8                                      0                 0
## 21-9                                      0                 0
## 28-1                                      0                 0
## 28-10                                     0                 0
## 28-11                                     0                 0
## 28-12                                     0                 0
## 28-13                                     0                 0
## 28-14                                    22                 0
## 28-15                                     0                 0
## 28-2                                      0                 0
## 28-3                                      0                 0
## 28-4                                      0                 0
## 28-5                                      0                 0
## 28-6                                      0                 0
## 28-7                                      0                 0
## 28-8                                      0                 0
## 28-9                                      0                 0
## 29-1                                      0                 0
## 29-10                                     0                 0
## 29-11                                     0                 0
## 29-12                                     0                 0
## 29-13                                     0                 0
## 29-14                                     0                 0
## 29-15                                     0                 0
## 29-2                                      0                 0
## 29-3                                      0                 0
## 29-4                                      0                 0
## 29-5                                      0                 0
## 29-6                                      0                 0
## 29-7                                      0                 0
## 29-8                                      0                 0
## 29-9                                      0                 0
##       Oscillatoria sp..36 Pseudolabrys sp.
## 20-10                   0                0
## 20-11                   0                0
## 20-12                   0                0
## 20-13                   0                0
## 20-14                   0                0
## 20-15                   0                0
## 20-2                    0                0
## 20-3                    0                0
## 20-4                    0                0
## 20-5                    0                0
## 20-6                    0                0
## 20-7                    0                0
## 20-8                    0                0
## 21-1                    0                0
## 21-10                   0                0
## 21-11                   0                0
## 21-12                   0                0
## 21-13                   0                0
## 21-14                   0                0
## 21-15                   0                0
## 21-2                    0                0
## 21-3                    0                0
## 21-4                    0                0
## 21-5                    0                0
## 21-6                    0                0
## 21-7                    0                0
## 21-8                    0                0
## 21-9                    0                0
## 28-1                    0                0
## 28-10                   0                0
## 28-11                   0                0
## 28-12                   0                0
## 28-13                   0                0
## 28-14                   0                0
## 28-15                   0                0
## 28-2                    0                0
## 28-3                    0                0
## 28-4                    0                0
## 28-5                    0                0
## 28-6                    0                0
## 28-7                    0                0
## 28-8                    0                0
## 28-9                    0                0
## 29-1                    0                0
## 29-10                   0                0
## 29-11                   0                0
## 29-12                   0                0
## 29-13                   0                0
## 29-14                   0                0
## 29-15                   0                0
## 29-2                    0                0
## 29-3                    0                0
## 29-4                    0                0
## 29-5                    0                0
## 29-6                    0                0
## 29-7                    0                0
## 29-8                    0                0
## 29-9                    0                0
##       Burkholderia-Caballeronia-Paraburkholderia sp. Sphingomonas sp..12
## 20-10                                              0                   0
## 20-11                                              0                   0
## 20-12                                              0                   0
## 20-13                                              0                   0
## 20-14                                              0                   0
## 20-15                                              0                   0
## 20-2                                               0                   0
## 20-3                                               0                   0
## 20-4                                               0                   0
## 20-5                                               0                   0
## 20-6                                               0                   0
## 20-7                                               0                   0
## 20-8                                               0                   0
## 21-1                                               0                   0
## 21-10                                              0                   0
## 21-11                                              0                   0
## 21-12                                              0                   0
## 21-13                                              0                   0
## 21-14                                              0                   0
## 21-15                                              0                   0
## 21-2                                               0                   0
## 21-3                                               0                   0
## 21-4                                               0                   0
## 21-5                                               0                   0
## 21-6                                               0                   0
## 21-7                                               0                   0
## 21-8                                               0                   0
## 21-9                                               0                   0
## 28-1                                               0                   0
## 28-10                                              0                   0
## 28-11                                              0                   0
## 28-12                                              0                   0
## 28-13                                              0                   0
## 28-14                                              0                   0
## 28-15                                              0                   0
## 28-2                                               0                   0
## 28-3                                               0                   0
## 28-4                                               0                   0
## 28-5                                               0                   0
## 28-6                                               0                   0
## 28-7                                               0                   0
## 28-8                                               0                   0
## 28-9                                               0                   0
## 29-1                                               0                   0
## 29-10                                              0                   0
## 29-11                                              0                   0
## 29-12                                              0                   0
## 29-13                                              0                   0
## 29-14                                              0                   0
## 29-15                                              0                   0
## 29-2                                               0                   0
## 29-3                                               0                   0
## 29-4                                               0                   0
## 29-5                                               0                   0
## 29-6                                               0                   0
## 29-7                                               0                   0
## 29-8                                               0                   0
## 29-9                                               0                   0
##       Sphingomonas sp..13 Aureimonas sp..12 Oscillatoria sp..37
## 20-10                   0                 0                   0
## 20-11                   0                 0                   0
## 20-12                   0                 0                   0
## 20-13                   0                 0                   0
## 20-14                   0                 0                   0
## 20-15                   0                 0                   0
## 20-2                    0                 0                   0
## 20-3                    0                 0                   0
## 20-4                    0                 0                   0
## 20-5                    0                 0                   0
## 20-6                    0                 0                   0
## 20-7                    0                 0                   0
## 20-8                    0                 0                   0
## 21-1                    0                 0                   0
## 21-10                   0                 0                   0
## 21-11                   0                 0                   0
## 21-12                   0                 0                   0
## 21-13                   0                 0                   0
## 21-14                   0                 0                   0
## 21-15                   0                 0                   0
## 21-2                    0                 0                   0
## 21-3                    0                 0                   0
## 21-4                    0                 0                   0
## 21-5                    0                 0                   0
## 21-6                    0                 0                   0
## 21-7                    0                 0                   0
## 21-8                    0                 0                   0
## 21-9                    0                 0                   0
## 28-1                    0                 0                   0
## 28-10                   0                 0                   0
## 28-11                   0                 0                   0
## 28-12                   0                 0                   0
## 28-13                   0                 0                   0
## 28-14                   0                 0                   0
## 28-15                   0                 0                   0
## 28-2                   20                 0                   0
## 28-3                    0                 0                   0
## 28-4                    0                 0                   0
## 28-5                    0                 0                   0
## 28-6                    0                 0                   0
## 28-7                    0                 0                   0
## 28-8                    0                 0                   0
## 28-9                    0                 0                   0
## 29-1                    0                 0                   0
## 29-10                   0                 0                   0
## 29-11                   0                 0                   0
## 29-12                   0                 0                   0
## 29-13                   0                 0                   0
## 29-14                   0                 0                   0
## 29-15                   0                 0                   0
## 29-2                    0                 0                   0
## 29-3                    0                 0                   0
## 29-4                    0                 0                   0
## 29-5                    0                 0                   0
## 29-6                    0                 0                   0
## 29-7                    0                 0                   0
## 29-8                    0                 0                   0
## 29-9                    0                 0                   0
##       Sphingomonas phyllosphaerae Oscillatoria sp..38 Aquamonas sp.
## 20-10                           0                   0             0
## 20-11                           0                   0             0
## 20-12                           0                   0             0
## 20-13                           0                   0             0
## 20-14                           0                   0             0
## 20-15                           0                   0             0
## 20-2                            0                   0             0
## 20-3                            0                   0             0
## 20-4                            0                   0             0
## 20-5                            0                   0             0
## 20-6                            0                   0             0
## 20-7                            0                   0             0
## 20-8                            0                   0             0
## 21-1                            0                   0             0
## 21-10                           0                   0             0
## 21-11                           0                   0             0
## 21-12                           0                   0             0
## 21-13                           0                   0             0
## 21-14                           0                   0             0
## 21-15                           0                   0             0
## 21-2                            0                   0             0
## 21-3                            0                   0             0
## 21-4                            0                   0             0
## 21-5                            0                   0            17
## 21-6                            0                   0             0
## 21-7                            0                   0             0
## 21-8                            0                   0             0
## 21-9                            0                   0             0
## 28-1                            0                   0             0
## 28-10                           0                   0             0
## 28-11                           0                   0             0
## 28-12                           0                   0             0
## 28-13                           0                   0             0
## 28-14                          18                   0             0
## 28-15                           0                   0             0
## 28-2                            0                   0             0
## 28-3                            0                   0             0
## 28-4                            0                   0             0
## 28-5                            0                  18             0
## 28-6                            0                   0             0
## 28-7                            0                   0             0
## 28-8                            0                   0             0
## 28-9                            0                   0             0
## 29-1                            0                   0             0
## 29-10                           0                   0             0
## 29-11                           0                   0             0
## 29-12                           0                   0             0
## 29-13                           0                   0             0
## 29-14                           0                   0             0
## 29-15                           0                   0             0
## 29-2                            0                   0             0
## 29-3                            0                   0             0
## 29-4                            0                   0             0
## 29-5                            0                   0             0
## 29-6                            0                   0             0
## 29-7                            0                   0             0
## 29-8                            0                   0             0
## 29-9                            0                   0             0
##       Hymenobacter norwichensis Aureimonas sp..13 Brevundimonas sp.
## 20-10                         0                 0                 0
## 20-11                         0                 0                 0
## 20-12                         0                 0                 0
## 20-13                         0                 0                 0
## 20-14                         0                 0                 0
## 20-15                         0                 0                 0
## 20-2                          0                 0                 0
## 20-3                          0                 0                 0
## 20-4                          0                 0                 0
## 20-5                          0                 0                 0
## 20-6                          0                 0                 0
## 20-7                          0                 0                 0
## 20-8                          0                 0                 0
## 21-1                          0                 0                 0
## 21-10                         0                 0                 0
## 21-11                         0                 0                 0
## 21-12                         0                 0                 0
## 21-13                         0                 0                 0
## 21-14                         0                 0                 0
## 21-15                         0                 0                 0
## 21-2                          0                 0                 0
## 21-3                          0                 0                 0
## 21-4                          0                 0                 0
## 21-5                          0                 0                 0
## 21-6                          0                 0                 0
## 21-7                          0                 0                 0
## 21-8                          0                 0                 0
## 21-9                          0                 0                 0
## 28-1                          0                 0                 0
## 28-10                         0                 0                 0
## 28-11                         0                 0                 0
## 28-12                         0                 0                 0
## 28-13                         0                 0                 0
## 28-14                         0                 0                 0
## 28-15                         0                 0                 0
## 28-2                          0                 0                 0
## 28-3                          0                 0                 0
## 28-4                          0                 0                 0
## 28-5                          0                 0                 0
## 28-6                          0                 0                 0
## 28-7                          0                 0                 0
## 28-8                          0                 0                 0
## 28-9                          0                 0                 0
## 29-1                          0                 0                 0
## 29-10                         0                 0                 0
## 29-11                         0                 0                 0
## 29-12                         0                 0                 0
## 29-13                         0                 0                 0
## 29-14                         0                 0                 0
## 29-15                         0                 0                 0
## 29-2                          0                 0                 0
## 29-3                          0                 0                 0
## 29-4                          0                 0                 0
## 29-5                          0                 0                 0
## 29-6                          0                 0                 0
## 29-7                          0                 0                 0
## 29-8                          6                 0                 0
## 29-9                          0                 0                 0
##       Escherichia-Shigella sp..7 Hymenobacter sp..9
## 20-10                          0                  0
## 20-11                          0                  0
## 20-12                          0                  0
## 20-13                          0                  0
## 20-14                          0                  0
## 20-15                          0                  0
## 20-2                           0                  0
## 20-3                           0                  0
## 20-4                           0                  0
## 20-5                           0                  0
## 20-6                           0                  0
## 20-7                           0                  0
## 20-8                           0                  0
## 21-1                           0                  0
## 21-10                          0                  0
## 21-11                          0                  0
## 21-12                          0                  0
## 21-13                          0                  0
## 21-14                          0                  0
## 21-15                          0                  0
## 21-2                           0                  0
## 21-3                           0                  0
## 21-4                           0                  0
## 21-5                           0                  0
## 21-6                           0                  0
## 21-7                           0                  0
## 21-8                           0                  0
## 21-9                           0                  0
## 28-1                           0                  0
## 28-10                          0                  0
## 28-11                          0                  0
## 28-12                          0                  0
## 28-13                          0                  0
## 28-14                          0                  0
## 28-15                          0                  0
## 28-2                           0                  0
## 28-3                           0                  0
## 28-4                           0                  0
## 28-5                           0                  0
## 28-6                           0                  0
## 28-7                           0                  0
## 28-8                           0                  0
## 28-9                           0                  0
## 29-1                           0                  0
## 29-10                          0                  0
## 29-11                          0                  0
## 29-12                          0                  0
## 29-13                          0                  0
## 29-14                          0                  0
## 29-15                          0                  0
## 29-2                           0                  0
## 29-3                           0                  0
## 29-4                           0                 15
## 29-5                           0                  0
## 29-6                           0                  0
## 29-7                           0                  0
## 29-8                           0                  0
## 29-9                           0                  0
##       Methylobacterium-Methylorubrum sp..49 Salana multivorans Roseomonas sp..2
## 20-10                                     0                  0                0
## 20-11                                     0                  0                0
## 20-12                                     0                  0                0
## 20-13                                     0                  0                0
## 20-14                                     0                  0                0
## 20-15                                     0                  0                0
## 20-2                                     14                  0                0
## 20-3                                      0                  0                0
## 20-4                                      0                  0                0
## 20-5                                      0                  0                0
## 20-6                                      0                  0                0
## 20-7                                      0                  0                0
## 20-8                                      0                  0                0
## 21-1                                      0                  0                0
## 21-10                                     0                 13                0
## 21-11                                     0                  0                0
## 21-12                                     0                  0                0
## 21-13                                     0                  0                0
## 21-14                                     0                  0                0
## 21-15                                     0                  0                0
## 21-2                                      0                  0                0
## 21-3                                      0                  0                0
## 21-4                                      0                  0                0
## 21-5                                      0                  0                0
## 21-6                                      0                  0                0
## 21-7                                      0                  0                0
## 21-8                                      0                  0                0
## 21-9                                      0                  0                7
## 28-1                                      0                  0                0
## 28-10                                     0                  0                0
## 28-11                                     0                  0                6
## 28-12                                     0                  0                0
## 28-13                                     0                  0                0
## 28-14                                     0                  0                0
## 28-15                                     0                  0                0
## 28-2                                      0                  0                0
## 28-3                                      0                  0                0
## 28-4                                      0                  0                0
## 28-5                                      0                  0                0
## 28-6                                      0                  0                0
## 28-7                                      0                  0                0
## 28-8                                      0                  0                0
## 28-9                                      0                  0                0
## 29-1                                      0                  0                0
## 29-10                                     0                  0                0
## 29-11                                     0                  0                0
## 29-12                                     0                  0                0
## 29-13                                     0                  0                0
## 29-14                                     0                  0                0
## 29-15                                     0                  0                0
## 29-2                                      0                  0                0
## 29-3                                      0                  0                0
## 29-4                                      0                  0                0
## 29-5                                      0                  0                0
## 29-6                                      0                  0                0
## 29-7                                      0                  0                0
## 29-8                                      0                  0                0
## 29-9                                      0                  0                0
##       Aurantimonas sp..3 Klenkia sp..1 Klenkia sp..2
## 20-10                  0             0             0
## 20-11                  0             0             0
## 20-12                  0             0             0
## 20-13                  0             0             0
## 20-14                  0             0             0
## 20-15                  0             0             0
## 20-2                   0             0             0
## 20-3                   0             0             0
## 20-4                   0             0             0
## 20-5                   0             0             0
## 20-6                   0             0             0
## 20-7                   0             0             0
## 20-8                   0             0             0
## 21-1                   0             0             0
## 21-10                  0             0             0
## 21-11                  0             0             0
## 21-12                  0             0             0
## 21-13                  0             0             0
## 21-14                  0             0             0
## 21-15                  0             0             0
## 21-2                   0             0             0
## 21-3                   0             0             0
## 21-4                   0             0             0
## 21-5                   0             0             0
## 21-6                   0             0             0
## 21-7                   0             0             0
## 21-8                   0             0             0
## 21-9                   0             0             0
## 28-1                   0             0             0
## 28-10                  0             0             0
## 28-11                  0             0             0
## 28-12                  0             0             0
## 28-13                  0             0             0
## 28-14                  0             0             0
## 28-15                  0             0             0
## 28-2                   0            11             0
## 28-3                   0             0             0
## 28-4                   0             0             0
## 28-5                   0             0             0
## 28-6                   0             0             0
## 28-7                   0             0             0
## 28-8                   0             0             0
## 28-9                   0             0             0
## 29-1                   0             0             0
## 29-10                  0             0             0
## 29-11                  0             0             0
## 29-12                  0             0             0
## 29-13                  0             0             0
## 29-14                  0             0             0
## 29-15                  0             0             0
## 29-2                   0             0             0
## 29-3                   0             0             0
## 29-4                   0             0             0
## 29-5                   0             0             0
## 29-6                   0             0             0
## 29-7                   0             0             0
## 29-8                   0             0             0
## 29-9                   0             0             0
Ambient=all.asvs %>% 
  map(filter, CO2 == "Control") %>% 
  map(select, -c(CO2)) %>% 
  map( column_to_rownames, "Sample")

# dumb way to do this, def better way to go about it. Stitch back together as a list
control.amb= c(Elevated, Ambient)
names(control.amb)<- c("elevated.its1", "elevated.its2", "elevated.16s", "amb.its1", "amb.its2", "amb.16s")

Make phyloseq objects of these networks

# make phyloseq otu tables 
otu= map(control.amb, otu_table, taxa_are_rows = F)

all.tax= all.tax %>% 
  map(.,~rownames_to_column(.x, var="seq"))

all.tax= map(all.tax, column_to_rownames,var= "uniqueID")

all.tax=map(all.tax, select, -"seq")


# duplicated these into elevated and ambient lists
Ambient.tax=all.tax 
Elevated.tax=all.tax 

# dumb way to do this, def better way to go about it. Stitch back together as a list
control.amb.tax= c(Elevated.tax, Ambient.tax)
names(control.amb.tax)<- c("elevated.its1", "elevated.its2", "elevated.16s", "amb.its1", "amb.its2", "amb.16s")


## make tax object

control.amb.tax= map(control.amb.tax, as.matrix)

TAX = map(control.amb.tax, tax_table)

# combine into phyloseq obj
phylo.all= map2(otu, TAX, phyloseq)

5.1 Create networks via SpeicEasi

From phyloseq obj

mapply(assign, names(phylo.all), phylo.all, MoreArgs=list(envir = globalenv()))
## $elevated.its1
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 397 taxa and 58 samples ]
## tax_table()   Taxonomy Table:    [ 397 taxa by 10 taxonomic ranks ]
## 
## $elevated.its2
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 1025 taxa and 58 samples ]
## tax_table()   Taxonomy Table:    [ 1025 taxa by 10 taxonomic ranks ]
## 
## $elevated.16s
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 190 taxa and 58 samples ]
## tax_table()   Taxonomy Table:    [ 190 taxa by 9 taxonomic ranks ]
## 
## $amb.its1
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 397 taxa and 59 samples ]
## tax_table()   Taxonomy Table:    [ 397 taxa by 10 taxonomic ranks ]
## 
## $amb.its2
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 1025 taxa and 59 samples ]
## tax_table()   Taxonomy Table:    [ 1025 taxa by 10 taxonomic ranks ]
## 
## $amb.16s
## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 190 taxa and 59 samples ]
## tax_table()   Taxonomy Table:    [ 190 taxa by 9 taxonomic ranks ]
#its1
# gen.se.elevated.its1 <- spiec.easi(elevated.its1, method='mb', lambda.min.ratio=1e-2, nlambda=100, pulsar.params=list(rep.num=50))
# 
# ## check stability 
# getStability(gen.se.elevated.its1) #0.045 that seems good enough
# 
# # 
# gen.se.amb.its1 <- spiec.easi(amb.its1, method='mb', lambda.min.ratio=1e-2, nlambda=100, pulsar.params=list(rep.num=50))
# 
# ## check stability 
# getStability(gen.se.amb.its1) #0.0426
# 
# #its2 
# pargs2 <- list(rep.num=50, seed=10010, ncores=4)
# 
# se.elevated.its2.bstars <- spiec.easi(elevated.its2, method='mb', lambda.min.ratio=1e-1, nlambda=100,
#                sel.criterion='bstars', pulsar.select=TRUE, pulsar.params=pargs2)
# 
# getStability(se.elevated.its2.bstars)  # 
# 
# save(se.elevated.its2.bstars, file = paste0(net.loc, "/spiec_nets_its2_ele.bstars.RData"))

# se.amb.its2.bstars <- spiec.easi(elevated.its2, method='mb', lambda.min.ratio=1e-1, nlambda=100,
#                sel.criterion='bstars', pulsar.select=TRUE, pulsar.params=pargs2)
# 
# getStability(se.amb.its2.bstars)  # 
# 


# bac
# gen.se.elevated.bac <- spiec.easi(elevated.16s, method='mb', lambda.min.ratio=1e-2, nlambda=100, pulsar.params=list(rep.num=50))

# changing lambda min ration because stability is kinda low. Alternative could be to raise nlambda but 100 was what the troublehooting guide recommended on the high side

# gen.se.elevated.bac <- spiec.easi(elevated.16s, method='mb', lambda.min.ratio=1e-1, nlambda=100, pulsar.params=list(rep.num=50))
# 
# getStability(gen.se.elevated.bac) #  0.0462359 better!
# gen.se.elevated.bac$select$stars$summary
# 
# # 
# gen.se.amb.bac <- spiec.easi(amb.16s, method='mb', lambda.min.ratio=1e-1, nlambda=100, pulsar.params=list(rep.num=50))
# 
# getStability(gen.se.amb.bac) # 0.0475

5.2 Save networks / work from a saved copy if necessary

#save(se.elevated.bac, se.amb.bac, file = paste0(net.loc, "/spiec_nets_16s.RData"))

#save(se.elevated.its1, se.amb.its1, file = paste0(net.loc, "spiec_nets_ITS1.RData"))

#save(se.amb.its2.bstars, se.elevated.its2.bstars, file = paste0(net.loc, "/spiec_nets_its2.RData"))


if(!"se.elevated.its1" %in% names(globalenv()))
{load(paste0(net.loc,"/spiec_nets_ITS1.RData"))}

if(!"se.elevated.bac" %in% names(globalenv()))
{load(paste0(net.loc,"/spiec_nets_16s.RData"))}

if(!"se.elevated.its2.bstars" %in% names(globalenv()))
{load(paste0(net.loc,"/spiec_nets_its2.RData"))}

5.3 Plot networks

ITS1

Elevated

# plot within phyloseq framework

its1.ele.mb <- adj2igraph(getRefit(se.elevated.its1),  vertex.attr=list(name=taxa_names(phylo.all$elevated.its1)))

p.its1.el= plot_network(its1.ele.mb, phylo.all$elevated.its1, type='taxa', color="Family.legend", label= NULL, point_size = 0.5, alpha= 0.5, line_alpha = 0.4, title = expression(paste("Elevated ", CO[2])))

# can do further ggplot functions 
p.its1.el= p.its1.el + 
  theme(legend.position = "none")

Ambient

its1.amb.mb <- adj2igraph(getRefit(se.amb.its1),  vertex.attr=list(name=taxa_names(phylo.all$amb.its1)))

p.its1.amb= plot_network(its1.amb.mb, phylo.all$amb.its1, type='taxa', color="Family.legend", label= NULL, point_size = 0.5, alpha= 0.5, line_alpha = 0.4, title = expression(paste("Ambient ", CO[2])))

p.its1.amb.2= p.its1.amb + 
  guides(color=guide_legend(ncol=2)) +
  theme(legend.position = "none") 

test= p.its1.amb + 
 labs(color = "") +
  theme_void(base_size = 10) + guides(color = guide_legend(override.aes = list(size=3)))
# Extract the legend. Returns a gtable
leg <- ggpubr::get_legend(test)

# Convert to a ggplot and print
leg.plot=as_ggplot(leg) 

Make a multiplot

p1= plot_grid(p.its1.amb.2, p.its1.el, labels = c('A', 'B'), hjust = 0.5)

p1.final= plot_grid(p1, NULL, leg.plot, ncol=1, align="v", rel_heights = c(1, 0, 1))

ggsave(paste0(output.loc,"/figureS3.jpg"),p1.final, height= 10)
## Saving 7 x 10 in image
ggsave(paste0(output.loc,"/figureS3.pdf"),p1.final, height= 10)
## Saving 7 x 10 in image

ITS2

# plot within phyloseq framework

el.its2.mb <- adj2igraph(getRefit(se.elevated.its2.bstars),  vertex.attr=list(name=taxa_names(phylo.all$elevated.its2)))

p.its2.el= plot_network(el.its2.mb, phylo.all$elevated.its2, type='taxa', color="Family.legend", label= NULL, point_size = 0.5, alpha= 0.5, line_alpha = 0.4, title = expression(paste("Elevated ", CO[2])))

# can do further ggplot functions 
p.its2.el= p.its2.el + 
  theme(legend.position = "none")
am.its2.mb <- adj2igraph(getRefit(se.amb.its2.bstars),  vertex.attr=list(name=taxa_names(phylo.all$amb.its2)))

p.its2.am= plot_network(am.its2.mb, phylo.all$amb.its2, type='taxa', color="Family.legend", label= NULL, point_size = 0.5, alpha= 0.5, line_alpha = 0.4, title = expression(paste("Ambient ", CO[2])))

# can do further ggplot functions 
p.its2.amb.2= p.its2.am + 
  guides(color=guide_legend(ncol=2)) +
  theme(legend.position = "none") 

#for the legend
leg.its2= p.its2.am + 
 labs(color = "") +
  theme_void(base_size = 10) + guides(color = guide_legend(override.aes = list(size=3)))
# Extract the legend. Returns a gtable
leg.its2 <- ggpubr::get_legend(leg.its2)

# Convert to a ggplot and print
leg.its2.plot=ggpubr::as_ggplot(leg.its2) 

Make a multiplot

p1.its2= plot_grid(p.its2.amb.2, p.its2.el, labels = c('A', 'B'), hjust = 0.5)

p1.its2.final= plot_grid(p1.its2, NULL, leg.its2.plot, ncol=1, align="v", rel_heights = c(1, 0, 1))

ggsave(paste0(output.loc,"/figure2.jpg"),p1.its2.final, height= 10)
## Saving 7 x 10 in image
ggsave(paste0(output.loc,"/figure2.pdf"),p1.its2.final, height= 10)
## Saving 7 x 10 in image

16s

#bac
bac.el.mb <- adj2igraph(getRefit(se.elevated.bac),  vertex.attr=list(name=taxa_names(phylo.all$elevated.16s)))

p.16s.el= plot_network(bac.el.mb, phylo.all$elevated.16s, type='taxa', color="Genus", point_size = 1, alpha= 0.5, line_alpha = 0.4, label= NULL, title = expression(paste("Elevated ", CO[2])))

p.16s.el= p.16s.el + theme(legend.position = "none")
bac.amb.mb <- adj2igraph(getRefit(se.amb.bac),  vertex.attr=list(name=taxa_names(phylo.all$amb.16s)))

p.16s.amb= plot_network(bac.amb.mb, phylo.all$amb.16s, type='taxa', color="Genus", point_size = 1, alpha= 0.5, line_alpha = 0.4, label= NULL, title = expression(paste("Ambient ", CO[2])))

p.16s.amb= p.16s.amb + theme(legend.position = "none")


#make legend edits and save to additional plot
bac.leg= p.16s.amb + 
 labs(color = "") +
  theme_void(base_size = 10) + guides(color = guide_legend(override.aes = list(size=3)))
# Extract the legend. Returns a gtable
bac.leg.extract <- ggpubr::get_legend(bac.leg)

# Convert to a ggplot and print
bac.leg.plot=as_ggplot(bac.leg.extract) 

Make a multiplot

bac1= plot_grid(p.16s.amb, p.16s.el, labels = c('A', 'B'), hjust = 0.5)

bac1.final= plot_grid(bac1, NULL, bac.leg.plot, ncol=1, align="v", rel_heights = c(1, 0, 1))

ggsave(paste0(output.loc,"/figure3.jpg"), bac1.final, height= 10)
## Saving 7 x 10 in image

5.4 Compare networks

Start with nets already made from spieceasi

# Make association matrix
# its1
assoMat.its1.el <- as.matrix(SpiecEasi::symBeta(getOptBeta(se.elevated.its1)))
assoMat.its1.am <- as.matrix(SpiecEasi::symBeta(getOptBeta(se.amb.its1)))

rownames(assoMat.its1.el) <- colnames(assoMat.its1.el) <- colnames(se.elevated.its1$est$data)
rownames(assoMat.its1.am) <- colnames(assoMat.its1.am) <- colnames(se.amb.its1$est$data)


net_its1_comp <- net_asso <- netConstruct(data = assoMat.its1.el, data2= assoMat.its1.am,
                         dataType = "condDependence",
                         sparsMethod = "none",
                         verbose = 0)

# its2
assoMat.its2.el <- as.matrix(SpiecEasi::symBeta(getOptBeta(se.elevated.its2.bstars)))
assoMat.its2.am <- as.matrix(SpiecEasi::symBeta(getOptBeta(se.amb.its2.bstars)))

rownames(assoMat.its2.el) <- colnames(assoMat.its2.el) <- colnames(se.elevated.its2.bstars$est$data)
rownames(assoMat.its2.am) <- colnames(assoMat.its2.am) <- colnames(se.amb.its2.bstars$est$data)


net_its2_comp <- net_asso <- netConstruct(data = assoMat.its2.el, data2= assoMat.its2.am,
                         dataType = "condDependence",
                         sparsMethod = "none",
                         verbose = 0)
# 16s
assoMat.bac.el <- as.matrix(SpiecEasi::symBeta(getOptBeta(se.elevated.bac)))
assoMat.bac.am <- as.matrix(SpiecEasi::symBeta(getOptBeta(se.amb.bac)))

rownames(assoMat.bac.el) <- colnames(assoMat.bac.el) <- colnames(se.elevated.bac$est$data)
rownames(assoMat.bac.am) <- colnames(assoMat.bac.am) <- colnames(se.amb.bac$est$data)


net_16s_comp <- net_asso <- netConstruct(data = assoMat.bac.el, data2= assoMat.bac.am,
                         dataType = "condDependence",
                         sparsMethod = "none",
                         verbose = 0)

5.5 Analyze network properties

Determine network properties for objects of class microNet

# Using eigenvector centrality as hub score

#its1
comp_its1_props1 <- netAnalyze(net_its1_comp, clustMethod = "cluster_fast_greedy", hubPar = "eigenvector")

plot(comp_its1_props1, groupNames = c("Elevated", "Ambient"))

outputits1.comp.props= summary(comp_its1_props1, groupNames = c("Elevated", "Ambient"))

#its2
comp_its2_props1 <- netAnalyze(net_its2_comp, clustMethod = "cluster_fast_greedy", hubPar = "eigenvector")

plot(comp_its2_props1, groupNames = c("Elevated", "Ambient"))

outputits2.comp.props= summary(comp_its2_props1, groupNames = c("Elevated", "Ambient"))

#16s
comp_16s_props1 <- netAnalyze(net_16s_comp, clustMethod = "cluster_fast_greedy", hubPar = "eigenvector")

plot(comp_16s_props1, groupNames = c("Elevated", "Ambient"))

output16s.comp.props= summary(comp_16s_props1, groupNames = c("Elevated", "Ambient"))

5.6 Quantitative network comparison

#its1
comp_its1 <- netCompare(comp_its1_props1, 
                          permTest = FALSE, 
                          verbose = FALSE,
                          seed = 123456)

sum.comp.its1=summary(comp_its1, 
        groupNames = c("Elevated CO2", "Ambient CO2"),
        showCentr = c("degree", "between", "closeness"), 
        numbNodes = 5)

#its2
comp_its2 <- netCompare(comp_its2_props1, 
                          permTest = FALSE, 
                          verbose = FALSE,
                          seed = 123456)

sum.comp.its2=summary(comp_its2, 
        groupNames = c("Elevated CO2", "Ambient CO2"),
        showCentr = c("degree", "between", "closeness"), 
        numbNodes = 5)

#16s
comp_bac <- netCompare(comp_16s_props1, 
                          permTest = FALSE, 
                          verbose = FALSE,
                          seed = 123456)

sum.comp.bac=summary(comp_bac, 
        groupNames = c("Elevated CO2", "Ambient CO2"),
        showCentr = c("degree", "between", "closeness"), 
        numbNodes = 5)

5.7 Make / output tables of relevant metrics

#hubs
hubs.bac= output16s.comp.props$hubs
hubs.bac$dataset <- "16s"

hubs.its1=outputits1.comp.props$hubs
hubs.its1$dataset <- "ITS1"

hubs.its2=outputits2.comp.props$hubs
hubs.its2$dataset <- "ITS2"

hubs= rbind(hubs.bac, hubs.its1)

hubs= rbind(hubs, hubs.its2)

write.csv(hubs, paste0(output.loc, "/hub.taxa.all.csv"))

# netcomp
netmets= sum.comp.its1$propdiffs[1:5,]
netmets$dataset <- "ITS1"
netmets=netmets %>% rownames_to_column("metric")

netmets.its2= sum.comp.its2$propdiffs[1:5,]
netmets.its2$dataset <- "ITS2"
netmets.its2=netmets.its2 %>% rownames_to_column("metric")

netmets.bac=sum.comp.bac$propdiffs[1:5,]
netmets.bac$dataset <- "16s"
netmets.bac= netmets.bac %>% rownames_to_column("metric")

netmets= rbind(netmets, netmets.bac)

netmets= rbind(netmets, netmets.its2)


colnames(netmets) <- c("metric", "Elevated CO2" , "Ambient CO2" ,"difference", "dataset")

write.csv(netmets, paste0(output.loc, "/net.metrics.all.csv"))

6 SUPPLEMENTAL

Compare ITS primers at the Genus and Family levels

its1.gen.unique=  unique(its1.tax$Genus)
its2.gen.unique=  unique(its2.tax$Genus)


its1.fam.unique=  unique(its1.tax$Family)
its2.fam.unique=  unique(its2.tax$Family)

#remove NA
its1.gen.unique= its1.gen.unique[!is.na(its1.gen.unique)]
its2.gen.unique= its2.gen.unique[!is.na(its2.gen.unique)]

its1.fam.unique= its1.fam.unique[!is.na(its1.fam.unique)]
its2.fam.unique= its2.fam.unique[!is.na(its2.fam.unique)]


# look at what is not shared
setdiff(its1.gen.unique, its2.gen.unique) #32 genera found in its1 not in its2
##  [1] "g__Pseudopithomyces"   "g__Paraboeremia"       "g__Mycosphaerella"    
##  [4] "g__Malassezia"         "g__Lectera"            "g__Neohelicomyces"    
##  [7] "g__Naganishia"         "g__Clitopilus"         "g__Schizophyllum"     
## [10] "g__Setoseptoria"       "g__Pyrenochaetopsis"   "g__Fomitopsis"        
## [13] "g__Sistotrema"         "g__Rhodotorula"        "g__Steccherinum"      
## [16] "g__Bullera"            "g__Exidia"             "g__Ceriporia"         
## [19] "g__Coprinellus"        "g__Roussoella"         "g__Paracylindrocarpon"
## [22] "g__Neocosmospora"      "g__Symmetrospora"      "g__Phaeopoacea"       
## [25] "g__Aspergillus"        "g__Vagicola"           "g__Neodevriesia"      
## [28] "g__Tomentella"         "g__Psilocybe"          "g__Pseudoophiobolus"  
## [31] "g__Bulleribasidium"    "g__Phomatospora"
setdiff(its2.gen.unique, its1.gen.unique) #33 general found in its2 not in its1
##  [1] "g__Cercospora"     "g__Didymocyrtis"   "g__Murispora"     
##  [4] "g__Blakeslea"      "g__Preussia"       "g__Gibellulopsis" 
##  [7] "g__Talaromyces"    "g__Xylodon"        "g__Subplenodomus" 
## [10] "g__Hymenoscyphus"  "g__Pestalotiopsis" "g__Phyllozyma"    
## [13] "g__Hyphoderma"     "g__Poitrasia"      "g__Paraphoma"     
## [16] "g__Plenodomus"     "g__Ceratobasidium" "g__Oliveonia"     
## [19] "g__Waitea"         "g__Sampaiozyma"    "g__Neptunomyces"  
## [22] "g__Trechispora"    "g__Hyphodermella"  "g__Flavodon"      
## [25] "g__Penicillium"    "g__Helicodendron"  "g__Laetisaria"    
## [28] "g__Sclerotrema"    "g__Moesziomyces"   "g__Neoroussoella" 
## [31] "g__Mycena"         "g__Kurtzmanomyces" "g__Ciliophora"
setdiff(its1.fam.unique, its2.fam.unique) #15 families found in its1 not in its2
##  [1] "f__Malasseziaceae"                   
##  [2] "f__Tubeufiaceae"                     
##  [3] "f__Entolomataceae"                   
##  [4] "f__Schizophyllaceae"                 
##  [5] "f__Cucurbitariaceae"                 
##  [6] "f__Fomitopsidaceae"                  
##  [7] "f__Lycoperdaceae"                    
##  [8] "f__Cantharellales_fam_Incertae_sedis"
##  [9] "f__Steccherinaceae"                  
## [10] "f__Bulleraceae"                      
## [11] "f__Exidiaceae"                       
## [12] "f__Symmetrosporaceae"                
## [13] "f__Thelephoraceae"                   
## [14] "f__Tilletiaceae"                     
## [15] "f__Phomatosporaceae"
setdiff(its2.fam.unique, its1.fam.unique) #20 families found in its2 not in its1
##  [1] "f__Amniculicolaceae"                 
##  [2] "f__Choanephoraceae"                  
##  [3] "f__Sporormiaceae"                    
##  [4] "f__Trichocomaceae"                   
##  [5] "f__Tetraplosphaeriaceae"             
##  [6] "f__Schizoporaceae"                   
##  [7] "f__Sporocadaceae"                    
##  [8] "f__Spiculogloeaceae"                 
##  [9] "f__Hyphodermataceae"                 
## [10] "f__Auriculariales_fam_Incertae_sedis"
## [11] "f__Trichomeriaceae"                  
## [12] "f__Xylariaceae"                      
## [13] "f__Chrysozymaceae"                   
## [14] "f__Hydnodontaceae"                   
## [15] "f__Phanerochaetaceae"                
## [16] "f__Lophiostomataceae"                
## [17] "f__Ustilaginaceae"                   
## [18] "f__Cordycipitaceae"                  
## [19] "f__Chionosphaeraceae"                
## [20] "f__Pezizomycotina_fam_Incertae_sedis"
# overlap
overlap.genus=intersect(its1.gen.unique,its2.gen.unique)
overlap.fam=intersect(its1.fam.unique,its2.fam.unique)

#proportion of taxa overlap
length(overlap.genus)/(length(overlap.genus) + length(setdiff(its1.gen.unique, its2.gen.unique))+ length(
setdiff(its2.gen.unique, its1.gen.unique))) # 47.81% taxa are shared at the genus level
## [1] 0.4758065
length(overlap.fam)/(length(overlap.fam) + length(setdiff(its1.fam.unique, its2.fam.unique))+ length(
setdiff(its2.fam.unique, its1.fam.unique))) # 55.70% taxa are shared at the fam level
## [1] 0.5512821

Venn diagrams

fam.list=list(ITS1= its1.fam.unique, ITS2= its2.fam.unique)
genera.list=list(ITS1= its1.gen.unique, ITS2= its2.gen.unique)

fam.venn=ggVennDiagram(fam.list) + 
  scale_fill_gradient(low="grey90",high = "red") +
  labs(title = "Taxa overlap by family")

genera.venn=ggVennDiagram(genera.list,) + scale_fill_gradient(low="grey90",high = "red") +
  labs(title = "Taxa overlap by genera")

ggsave(paste0(output.loc,"/figureS1.fam.fenn.pdf"), fam.venn)
## Saving 8 x 5 in image
ggsave(paste0(output.loc,"/figureS2.genera.fenn.pdf"), genera.venn)
## Saving 8 x 5 in image